Sample records for evolutionarily conserved transcriptional

During Drosophila eye development, differentiation initiates in the posterior region of the eye disk and progresses anteriorly as a wave marked by the morphogenetic furrow (MF), which demarcates the boundary between anterior undifferentiated cells and posterior differentiated photoreceptors. However, the mechanism underlying the regulation of gene expression immediately before the onset of differentiation remains unclear. Here, we show that Apontic (Apt), which is an evolutionarilyconservedtranscription factor, is expressed in the differentiating cells posterior to the MF. Moreover, it directly induces the expression of cyclin E and is also required for the G1-to-S phase transition, which is known to be essential for the initiation of cell differentiation at the MF. These observations identify a pathway crucial for eye development, governed by a mechanism in which Cyclin E promotes the G1-to-S phase transition when regulated by Apt.

Full Text Available T-type calcium channels operate within tightly regulated biophysical constraints for supporting rhythmic firing in the brain, heart and secretory organs of invertebrates and vertebrates. The snail T-type gene, LCa(v3 from Lymnaea stagnalis, possesses alternative, tandem donor splice sites enabling a choice of a large exon 8b (201 aa or a short exon 25c (9 aa in cytoplasmic linkers, similar to mammalian homologs. Inclusion of optional 25c exons in the III-IV linker of T-type channels speeds up kinetics and causes hyperpolarizing shifts in both activation and steady-state inactivation of macroscopic currents. The abundant variant lacking exon 25c is the workhorse of embryonic Ca(v3 channels, whose high density and right-shifted activation and availability curves are expected to increase pace-making and allow the channels to contribute more significantly to cellular excitation in prenatal tissue. Presence of brain-enriched, optional exon 8b conserved with mammalian Ca(v3.1 and encompassing the proximal half of the I-II linker, imparts a ~50% reduction in total and surface-expressed LCa(v3 channel protein, which accounts for reduced whole-cell calcium currents of +8b variants in HEK cells. Evolutionarilyconserved optional exons in cytoplasmic linkers of Ca(v3 channels regulate expression (exon 8b and a battery of biophysical properties (exon 25c for tuning specialized firing patterns in different tissues and throughout development.

Full Text Available Color variation provides the opportunity to investigate the genetic basis of evolution and selection. Reptiles are less studied than mammals. Comparative genomics approaches allow for knowledge gained in one species to be leveraged for use in another species. We describe a comparative vertebrate analysis of conserved regulatory modules in pythons aimed at assessing bioinformatics evidence that transcription factors important in mammalian pigmentation phenotypes may also be important in python pigmentation phenotypes. We identified 23 python orthologs of mammalian genes associated with variation in coat color phenotypes for which we assessed the extent of pairwise protein sequence identity between pythons and mouse, dog, horse, cow, chicken, anole lizard, and garter snake. We next identified a set of melanocyte/pigment associated transcription factors (CREB, FOXD3, LEF-1, MITF, POU3F2, and USF-1 that exhibit relatively conserved sequence similarity within their DNA binding regions across species based on orthologous alignments across multiple species. Finally, we identified 27 evolutionarilyconserved clusters of transcription factor binding sites within ~200-nucleotide intervals of the 1500-nucleotide upstream regions of AIM1, DCT, MC1R, MITF, MLANA, OA1, PMEL, RAB27A, and TYR from Python bivittatus. Our results provide insight into pigment phenotypes in pythons.

Color variation provides the opportunity to investigate the genetic basis of evolution and selection. Reptiles are less studied than mammals. Comparative genomics approaches allow for knowledge gained in one species to be leveraged for use in another species. We describe a comparative vertebrate analysis of conserved regulatory modules in pythons aimed at assessing bioinformatics evidence that transcription factors important in mammalian pigmentation phenotypes may also be important in python pigmentation phenotypes. We identified 23 python orthologs of mammalian genes associated with variation in coat color phenotypes for which we assessed the extent of pairwise protein sequence identity between pythons and mouse, dog, horse, cow, chicken, anole lizard, and garter snake. We next identified a set of melanocyte/pigment associated transcription factors (CREB, FOXD3, LEF-1, MITF, POU3F2, and USF-1) that exhibit relatively conserved sequence similarity within their DNA binding regions across species based on orthologous alignments across multiple species. Finally, we identified 27 evolutionarilyconserved clusters of transcription factor binding sites within ~200-nucleotide intervals of the 1500-nucleotide upstream regions of AIM1, DCT, MC1R, MITF, MLANA, OA1, PMEL, RAB27A, and TYR from Python bivittatus. Our results provide insight into pigment phenotypes in pythons. PMID:27698666

Comparison of human sequences with the DNA of other mammals is an excellent means of identifying functional elements in the human genome. Here we describe the utility of high-density oligonucleotide arrays as a rapid approach for comparing human sequences with the DNA of multiple species whose sequences are not presently available. High-density arrays representing approximately 22.5 Mb of nonrepetitive human chromosome 21 sequence were synthesized and then hybridized with mouse and dog DNA to identify sequences conserved between humans and mice (human-mouse elements) and between humans and dogs (human-dog elements). Our data show that sequence comparison of multiple species provides a powerful empiric method for identifying actively conserved elements in the human genome. A large fraction of these evolutionarilyconserved elements are present in regions on chromosome 21 that do not encode known genes.

Full Text Available There is considerable debate about the functionality of long non-coding RNAs (lncRNAs. Lack of sequence conservation has been used to argue against functional relevance. We investigated antisense lncRNAs, called COOLAIR, at the A. thaliana FLC locus and experimentally determined their secondary structure. The major COOLAIR variants are highly structured, organized by exon. The distally polyadenylated transcript has a complex multi-domain structure, altered by a single non-coding SNP defining a functionally distinct A. thaliana FLC haplotype. The A. thaliana COOLAIR secondary structure was used to predict COOLAIR exons in evolutionarily divergent Brassicaceae species. These predictions were validated through chemical probing and cloning. Despite the relatively low nucleotide sequence identity, the structures, including multi-helix junctions, show remarkable evolutionary conservation. In a number of places, the structure is conserved through covariation of a non-contiguous DNA sequence. This structural conservation supports a functional role for COOLAIR transcripts rather than, or in addition to, antisense transcription.

In recent years, the transcription factor ABI4 has emerged as an important node of integration for externaland internal signals such as nutrient status and hormone signaling that modulates critical transitions during the growthand development of plants. For this reason, understanding the mechanism of action and regulation of this protein rep-resents an important step towards the elucidation of crosstalk mechanisms in plants. However, this understanding hasbeen hindered due to the negligible levels of this protein as a result of multiple posttranscriptional regulations. To betterunderstand the function and regulation of the ABI4 protein in this work, we performed a functional analysis of severalevolutionarily conserved motifs. Based on these conserved motifs, we identified ortholog genes of ABI4 in differentplant species. The functionality of the putative ortholog from Theobroma cacao was demonstrated in transient expres-sion assays and in complementation studies in plants. The function of the highly conserved motifs was analyzed aftertheir deletion or mutagenesis in the Arabidopsis ABI4 sequence using mesophyll protoplasts. This approach permitted usto immunologically detect the ABI4 protein and identify some of the mechanisms involved in its regulation. We identi-fied sequences required for the nuclear localization （AP2-associated motif） as well as those for transcriptional activationfunction （LRP motif）. Moreover, this approach showed that the protein stability of this transcription factor is controlledthrough protein degradation and subcellular localization and involves the AP2-associated and the PEST motifs. We dem-onstrated that the degradation of ABI4 protein through the PEST motif is mediated by the 26S proteasome in responseto changes in the sugar levels.

Full Text Available The question of a potential biological sexual signature in the human brain is a heavily disputed subject. In order to provide further insight into this issue, we used an evolutionary approach to identify genes with sex differences in brain expression level among primates. We reasoned that expression patterns important to uphold key male and female characteristics may be conserved during evolution. We selected cortex for our studies because this specific brain region is responsible for many higher behavioral functions. We compared gene expression profiles in the occipital cortex of male and female humans (Homo sapiens, a great ape and cynomolgus macaques (Macaca fascicularis, an old world monkey, two catarrhine species that show abundant morphological sexual dimorphism, as well as in common marmosets (Callithrix Jacchus, a new world monkey which are relatively sexually monomorphic. We identified hundreds of genes with sex-biased expression patterns in humans and macaques, while fewer than ten were differentially expressed between the sexes in marmosets. In primates, a general rule is that many of the morphological and behavioral sexual dimorphisms seen in polygamous species, such as macaques, are typically less pronounced in monogamous species such as the marmosets. Our observations suggest that this correlation may also be reflected in the extent of sex-biased gene expression in the brain. We identified 85 genes with common sex-biased expression, in both human and macaque and 2 genes, X inactivation-specific transcript (XIST and Heat shock factor binding protein 1 (HSBP1, that were consistently sex-biased in the female direction in human, macaque, and marmoset. These observations imply a conserved signature of sexual gene expression dimorphism in cortex of primates. Further, we found that the coding region of female-biased genes is more evolutionarily constrained compared to the coding region of both male-biased and non sex-biased brain

Legionella pneumophila proliferates within various protists and metazoan cells, where a cadre of ∼300 effectors is injected into the host cell by the defect in organelle trafficking/intracellular multiplication (Dot/Icm) type IVB translocation system. Interkingdom horizontal gene transfer of genes of protists and their subsequent convergent evolution to become translocated effectors has probably enabled L. pneumophila to adapt to the intracellular life within various protists and metazoan cells through exploitation of evolutionarily eukaryotic processes, such as endoplasmic reticulum-to-Golgi vesicle traffic, phosphoinositol metabolism, AMPylation, deAMPylation, prenylation, polyubiquitination, proteasomal degradation and cytosolic amino- and oligo-peptidases. This is highlighted by the ankyrin B (AnkB) F-box effector that exploits multiple conserved eukaryotic machineries to generate high levels of free amino acids as sources of carbon and energy essential for intracellular proliferation in protists and metazoan cells and for manifestation of pulmonary disease in mammals.

Full Text Available Abstract Background The strong linkage disequilibrium (LD recently found in genic or exonic regions of the human genome demonstrated that LD can be increased by evolutionary mechanisms that select for functionally important loci. This suggests that LD might be stronger in regions conserved among species than in non-conserved regions, since regions exposed to natural selection tend to be conserved. To assess this hypothesis, we used genome-wide polymorphism data from the HapMap project and investigated LD within DNA sequences conserved between the human and mouse genomes. Results Unexpectedly, we observed that LD was significantly weaker in conserved regions than in non-conserved regions. To investigate why, we examined sequence features that may distort the relationship between LD and conserved regions. We found that interspersed repeats, and not other sequence features, were associated with the weak LD tendency in conserved regions. To appropriately understand the relationship between LD and conserved regions, we removed the effect of repetitive elements and found that the high degree of sequence conservation was strongly associated with strong LD in coding regions but not with that in non-coding regions. Conclusion Our work demonstrates that the degree of sequence conservation does not simply increase LD as predicted by the hypothesis. Rather, it implies that purifying selection changes the polymorphic patterns of coding sequences but has little influence on the patterns of functional units such as regulatory elements present in non-coding regions, since the former are generally restricted by the constraint of maintaining a functional protein product across multiple exons while the latter may exist more as individually isolated units.

Full Text Available The evolution of enzymes affects how well a species can adapt to new environmental conditions. During enzyme evolution, certain aspects of molecular function are conserved while other aspects can vary. Aspects of function that are more difficult to change or that need to be reused in multiple contexts are often conserved, while those that vary may indicate functions that are more easily changed or that are no longer required. In analogy to the study of conservation patterns in enzyme sequences and structures, we have examined the patterns of conservation and variation in enzyme function by analyzing graph isomorphisms among enzyme substrates of a large number of enzyme superfamilies. This systematic analysis of substrate substructures establishes the conservation patterns that typify individual superfamilies. Specifically, we determined the chemical substructures that are conserved among all known substrates of a superfamily and the substructures that are reacting in these substrates and then examined the relationship between the two. Across the 42 superfamilies that were analyzed, substantial variation was found in how much of the conserved substructure is reacting, suggesting that superfamilies may not be easily grouped into discrete and separable categories. Instead, our results suggest that many superfamilies may need to be treated individually for analyses of evolution, function prediction, and guiding enzyme engineering strategies. Annotating superfamilies with these conserved and reacting substructure patterns provides information that is orthogonal to information provided by studies of conservation in superfamily sequences and structures, thereby improving the precision with which we can predict the functions of enzymes of unknown function and direct studies in enzyme engineering. Because the method is automated, it is suitable for large-scale characterization and comparison of fundamental functional capabilities of both characterized

Full Text Available Plants have a remarkable ability to react to seasonal changes by synchronizing life-cycle transitions with environmental conditions. We addressed the question of how transcriptional re-programming occurs in response to an environmental cue that triggers the major life cycle transition from seed dormancy to germination and seedling growth. We elucidated an important mechanistic aspect of this process by following the chromatin dynamics of key regulatory genes with a focus on the two antagonistic marks, H3K4me3 and H3K27me3. Histone methylation patterns of major dormancy regulators changed during the transition to germination and seedling growth. We observed a switch from H3K4me3 and high transcription levels to silencing by the repressive H3K27me3 mark when dormancy was broken through exposure to moist chilling, underscoring that a functional PRC2 complex is necessary for this transition. Moreover, this reciprocal regulation by H3K4me3 and H3K27me3 is evolutionarilyconserved from gymnosperms to angiosperms.

higher fractions of the more compact Drosophila melanogaster (37%-53%), Caenorhabditis elegans (18%-37%), and Saccharaomyces cerevisiae (47%-68%) genomes. From yeasts to vertebrates, in order of increasing genome size and general biological complexity, increasing fractions of conserved bases are found...... species of Drosophila and Anopheles gambiae), two species of Caenorhabditis, and seven species of Saccharomyces. Conserved elements were identified with a computer program called phastCons, which is based on a two-state phylogenetic hidden Markov model (phylo-HMM). PhastCons works by fitting a phylo...

Diurnal patterns of gene transcription are often conferred by complex interactions between circadian clock control and acute responses to environmental cues. Arabidopsis thaliana GIGANTEA (GI) contributes to photoperiodic flowering, circadian clock control, and photoreceptor signaling, and its transcription is regulated by the circadian clock and light. We used phylogenetic shadowing to identify three evolutionarily constrained regions (conserved regulatory modules [CRMs]) within the GI promoter and show that CRM2 is sufficient to confer a similar transcriptional pattern as the full-length promoter. Dissection of CRM2 showed that one subfragment (CRM2-A) contributes light inducibility, while another (CRM2-B) exhibits a diurnal response. Mutational analysis showed that three ABA RESPONSE ELEMENT LIKE (ABREL) motifs in CRM2-A and three EVENING ELEMENTs (EEs) in CRM2-B are essential in combination to confer a high amplitude diurnal pattern of expression. Genome-wide analysis identified characteristic spacing patterns of EEs and 71 A. thaliana promoters containing three EEs. Among these promoters, that of FLAVIN BINDING KELCH REPEAT F-BOX1 was analyzed in detail and shown to harbor a CRM functionally related to GI CRM2. Thus, combinatorial interactions among EEs and ABRELs confer diurnal patterns of transcription via an evolutionarilyconserved module present in GI and other evening-expressed genes. PMID:25361953

The Hippo pathway represses YAP oncoprotein activity through phosphorylation by LATS kinases. Although variety of upstream components has been found to participate in the Hippo pathway, the existence and function of negative feedback has remained uncertain. We found that activated YAP, together with TEAD transcription factors, directly induces transcription of LATS2, but not LATS1, to form a negative feedback loop. We also observed increased mRNA levels of Hippo upstream components upon YAP activation. To reveal the physiological role of this negative feedback regulation, we deleted Lats2 or Lats1 in the liver-specific Sav1-knockout mouse model which develops a YAP-induced tumor. Additional deletion of Lats2 severely enhanced YAP-induced tumorigenic phenotypes in a liver specific Sav1 knock-out mouse model while additional deletion of Lats1 mildly affected the phenotype. Only Sav1 and Lats2 double knock-down cells formed larger colonies in soft agar assay, thereby recapitulating accelerated tumorigenesis seen in vivo. Importantly, this negative feedback is evolutionarilyconserved, as Drosophila Yorkie (YAP ortholog) induces transcription of Warts (LATS2 ortholog) with Scalloped (TEAD ortholog). Collectively, we demonstrated the existence and function of an evolutionarilyconserved negative feedback mechanism in the Hippo pathway, as well as the functional difference between LATS1 and LATS2 in regulation of YAP.

Full Text Available Knowledge of the functional cis-regulatory elements that regulate constitutive and alternative pre-mRNA splicing is fundamental for biology and medicine. Here we undertook a genome-wide comparative genomics approach using available mammalian genomes to identify conserved intronic splicing regulatory elements (ISREs. Our approach yielded 314 ISREs, and insertions of ~70 ISREs between competing splice sites demonstrated that 84% of ISREs altered 5' and 94% altered 3' splice site choice in human cells. Consistent with our experiments, comparisons of ISREs to known splicing regulatory elements revealed that 40%-45% of ISREs might have dual roles as exonic splicing silencers. Supporting a role for ISREs in alternative splicing, we found that 30%-50% of ISREs were enriched near alternatively spliced (AS exons, and included almost all known binding sites of tissue-specific alternative splicing factors. Further, we observed that genes harboring ISRE-proximal exons have biases for tissue expression and molecular functions that are ISRE-specific. Finally, we discovered that for Nova1, neuronal PTB, hnRNP C, and FOX1, the most frequently occurring ISRE proximal to an alternative conserved exon in the splicing factor strongly resembled its own known RNA binding site, suggesting a novel application of ISRE density and the propensity for splicing factors to auto-regulate to associate RNA binding sites to splicing factors. Our results demonstrate that ISREs are crucial building blocks in understanding general and tissue-specific AS regulation and the biological pathways and functions regulated by these AS events.

Slight shifts in arrangement within biological photonic nanostructures can produce large color differences, and sexual selection often leads to high color diversity in clades with structural colors. We use phylogenetic reconstruction, electron microscopy, spectrophotometry, and optical modeling to show an opposing pattern of nanostructural diversification accompanied by unusual conservation of blue color in tarantulas (Araneae: Theraphosidae). In contrast to other clades, blue coloration in phylogenetically distant tarantulas peaks within a narrow 20-nm region around 450 nm. Both quasi-ordered and multilayer nanostructures found in different tarantulas produce this blue color. Thus, even within monophyletic lineages, tarantulas have evolved strikingly similar blue coloration through divergent mechanisms. The poor color perception and lack of conspicuous display during courtship of tarantulas argue that these colors are not sexually selected. Therefore, our data contrast with sexual selection that typically produces a diverse array of colors with a single structural mechanism by showing that natural selection on structural color in tarantulas resulted in convergence on similar color through diverse structural mechanisms.

Animals have developed the means for supporting complex and dynamic consortia of microorganisms during their life cycle. A transcendent view of vertebrate biology therefore requires an understanding of the contributions of these indigenous microbial communities to host development and adult physiology. These contributions are most obvious in the gut, where studies of gnotobiotic mice have disclosed that the microbiota affects a wide range of biological processes, including nutrient processing and absorption, development of the mucosal immune system, angiogenesis, and epithelial renewal. The zebrafish (Danio rerio) provides an opportunity to investigate the molecular mechanisms underlying these interactions through genetic and chemical screens that take advantage of its transparency during larval and juvenile stages. Therefore, we developed methods for producing and rearing germ-free zebrafish through late juvenile stages. DNA microarray comparisons of gene expression in the digestive tracts of 6 days post fertilization germ-free, conventionalized, and conventionally raised zebrafish revealed 212 genes regulated by the microbiota, and 59 responses that are conserved in the mouse intestine, including those involved in stimulation of epithelial proliferation, promotion of nutrient metabolism, and innate immune responses. The microbial ecology of the digestive tracts of conventionally raised and conventionalized zebrafish was characterized by sequencing libraries of bacterial 16S rDNA amplicons. Colonization of germ-free zebrafish with individual members of its microbiota revealed the bacterial species specificity of selected host responses. Together, these studies establish gnotobiotic zebrafish as a useful model for dissecting the molecular foundations of host-microbial interactions in the vertebrate digestive tract. PMID:15070763

Full Text Available Abstract Background Clusterin associated protein 1 (CLUAP1 was initially characterized as a protein that interacts with clusterin, and whose gene is frequently upregulated in colon cancer. Although the consequences of these observations remain unclear, research of CLUAP1 homologs in C. elegans and zebrafish indicates that it is needed for cilia assembly and maintenance in these models. To begin evaluating whether Cluap1 has an evolutionarilyconserved role in cilia in mammalian systems and to explore the association of Cluap1 with disease pathogenesis and developmental abnormalities, we generated Cluap1 mutant mice. Methods Cluap1 mutant embryos were generated and examined for gross morphological and anatomical defects using light microscopy. Reverse transcription PCR, β-galactosidase staining assays, and immunofluorescence analysis were used to determine the expression of the gene and localization of the protein in vivo and in cultured cell lines. We also used immunofluorescence analysis and qRT-PCR to examine defects in the Sonic hedgehog signaling pathway in mutant embryos. Results Cluap1 mutant embryos die in mid-gestation, indicating that it is necessary for proper development. Mutant phenotypes include a failure of embryonic turning, an enlarged pericardial sac, and defects in neural tube development. Consistent with the diverse phenotypes, Cluap1 is widely expressed. Furthermore, the Cluap1 protein localizes to primary cilia, and mutant embryos were found to lack cilia at embryonic day 9.5. The phenotypes observed in Cluap1 mutant mice are indicative of defects in Sonic hedgehog signaling. This was confirmed by analyzing hedgehog signaling activity in Cluap1 mutants, which revealed that the pathway is repressed. Conclusions These data indicate that the function of Cluap1 is evolutionarilyconserved with regard to ciliogenesis. Further, the results implicate mammalian Cluap1 as a key regulator of hedgehog signaling and as an

Plant breeding relies on creation of novel allelic combinations for desired traits. Identification and utilization of beneficial alleles, rare alleles and evolutionarilyconserved genes in the germplasm (referred to as 'hidden' genes) provide an effective approach to achieve this goal. Here we show that a chemically induced null mutation in an evolutionarilyconserved gene, FUWA, alters multiple important agronomic traits in rice, including panicle architecture, grain shape and grain weight. FUWA encodes an NHL domain-containing protein, with preferential expression in the root meristem, shoot apical meristem and inflorescences, where it restricts excessive cell division. Sequence analysis revealed that FUWA has undergone a bottleneck effect, and become fixed in landraces and modern cultivars during domestication and breeding. We further confirm a highly conserved role of FUWA homologs in determining panicle architecture and grain development in rice, maize and sorghum through genetic transformation. Strikingly, knockdown of the FUWA transcription level by RNA interference results in an erect panicle and increased grain size in both indica and japonica genetic backgrounds. This study illustrates an approach to create new germplasm with improved agronomic traits for crop breeding by tapping into evolutionary conserved genes.

Full Text Available Abstract Background Factors that affect flowering vary among different plant species, and in the grasses in particular the exact mechanism behind this transition is not fully understood. The brown midrib (bm mutants of maize (Zea mays L., which have altered cell wall composition, have different flowering dynamics compared to their wild-type counterparts. This is indicative of a link between cell wall biogenesis and flowering. In order to test whether this relationship also exists in other grasses, the flowering dynamics in sorghum (Sorghum bicolor (L. Moench were investigated. Sorghum is evolutionarily closely related to maize, and a set of brown midrib (bmr mutants similar to the maize bm mutants is available, making sorghum a suitable choice for study in this context. Results We compared the flowering time (time to half-bloom of several different bmr sorghum lines and their wild-type counterparts. This revealed that the relationship between cell wall composition and flowering was conserved in sorghum. Specifically, the mutant bmr7 flowered significantly earlier than the corresponding wild-type control, whereas the mutants bmr2, bmr4, bmr6, bmr12, and bmr19 flowered later than their wild-type controls. Conclusion The change in flowering dynamics in several of the brown midrib sorghum lines provides evidence for an evolutionarilyconserved mechanism that links cell wall biosynthesis to flowering dynamics. The availability of the sorghum bmr mutants expands the germplasm available to investigate this relationship in further detail.

Macroautophagy/autophagy is a catabolic process that is essential for cellular homeostasis. Studies on autophagic degradation of cytoplasmic components have generated interest in nuclear autophagy. Although its mechanisms and roles have remained elusive, tremendous progress has been made toward understanding nuclear autophagy. Nuclear autophagy is evolutionarilyconserved in eukaryotes that may target various nuclear components through a series of processes, including nuclear sensing, nuclear export, autophagic substrate encapsulation and autophagic degradation in the cytoplasm. However, the molecular processes and regulatory mechanisms involved in nuclear autophagy remain largely unknown. Numerous studies have highlighted the importance of nuclear autophagy in physiological and pathological processes such as cancer. This review focuses on current advances in nuclear autophagy and provides a summary of its research history and landmark discoveries to offer new perspectives.

Abstract The olfactory cortex is part of the mammalian cerebral cortex together with the neocortex and the hippocampus. It receives direct input from the olfactory bulbs and participates in odor discrimination, association, and learning (Bekkers and Suzuki, 2013). It is thought to be an evolutionarilyconserved paleocortex, which shares common characteristics with the three-layered general cortex of reptiles (Aboitiz et al., 2002). The olfactory cortex has been studied as a “simple model” to address sensory processing, though little is known about its precise cell origin, diversity, and identity. While the development and the cellular diversity of the six-layered neocortex are increasingly understood, the olfactory cortex remains poorly documented in these aspects. Here is a review of current knowledge of the development and organization of the olfactory cortex, keeping the analogy with those of the neocortex. The comparison of olfactory cortex and neocortex will allow the opening of evolutionary perspectives on cortical development.

The Toll/Toll-like receptor (TLR) signaling pathway has an important role in the innate immunity of animals. Evolutionarilyconserved signaling intermediate in Toll pathways (ECSIT) is a protein that functions as an adaptor protein for the Toll/TLR and bone morphogenetic protein signaling pathways. ECSIT is also a key component in the macrophage bactericidal activity of mammals. However, the function of ECSIT in crustaceans remains unclear. In this study, we cloned and identified a functional ECSIT homologue, MjECSIT 1, from kuruma shrimp Marsupenaeus japonicus. The complementary DNA of MjEcsit 1 is 1442 base pairs long, with an open reading frame of 1221 base pairs that encodes a 407-residue polypeptide. Transcripts of MjEcsit 1 are detected in hemocytes, gills, hepatopancreas, stomach, heart, intestines, testes, and ovaries. Such transcripts are upregulated by Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Vibrio anguillarum) injections. The knockdown of MjEcsit 1 by double-stranded RNA injection increases the sensitivity of M. japonicus to S. aureus challenge and weakens the bacterial clearance ability of M. japonicus in vivo. In addition, suppressing MjEcsit 1 restrains the upregulation of two anti-lipopolysaccharide factors by S. aureus injection. The results indicate that MjECSIT 1 is important in the antibacterial immunity of M. japonicus.

Full Text Available During brain development, neural stem cells (NSCs receive on-or-off signals important for regulating their amplification and reaching adequate neuron density. However, how a coordinated regulation of intracellular pathways and genetic programs is achieved has remained elusive. Here, we found that the embryonic (e CSF contains 10¹² nanoparticles/ml (77 nm diameter, some of which were identified as exosome nanovesicles that contain evolutionarilyconserved molecules important for coordinating intracellular pathways. eCSF nanovesicles collected from rodent and human embryos encapsulate protein and microRNA components of the insulin-like growth factor (IGF signaling pathway. Supplementation of eCSF nanovesicles to a mixed culture containing eNSCs activated the IGF-mammalian target of rapamycin complex 1 (mTORC1 pathway in eNSCs and expanded the pool of proliferative eNSCs. These data show that the eCSF serves as a medium for the distribution of nanovesicles, including exosomes, and the coordinated transfer of evolutionary conserved molecules that regulate eNSC amplification during corticogenesis.

Non-coding RNAs (ncRNAs) have diverse essential biological functions in all organisms, and in eukaryotes, two such classes of ncRNAs are the small nucleolar (sno) and small nuclear (sn) RNAs. In this study, we have identified and characterized a collection of sno and snRNAs in Giardia lamblia, by exploiting our discovery of a conserved 12 nt RNA processing sequence motif found in the 3′ end regions of a large number of G. lamblia ncRNA genes. RNA end mapping and other experiments indicate the motif serves to mediate ncRNA 3′ end formation from mono- and di-cistronic RNA precursor transcripts. Remarkably, we find the motif is also utilized in the processing pathway of all four previously identified trans-spliced G. lamblia introns, revealing a common RNA processing pathway for ncRNAs and trans-spliced introns in this organism. Motif sequence conservation then allowed for the bioinformatic and experimental identification of additional G. lamblia ncRNAs, including new U1 and U6 spliceosomal snRNA candidates. The U6 snRNA candidate was then used as a tool to identity novel U2 and U4 snRNAs, based on predicted phylogenetically conserved snRNA–snRNA base-pairing interactions, from a set of previously identified G. lamblia ncRNAs without assigned function. The Giardia snRNAs retain the core features of spliceosomal snRNAs but are sufficiently evolutionarily divergent to explain the difficulties in their identification. Most intriguingly, all of these snRNAs show structural features diagnostic of U2-dependent/major and U12-dependent/minor spliceosomal snRNAs. PMID:23019220

Tubulin polymerization promoting proteins (TPPPs) constitute a eukaryotic protein family. There are three TPPP paralogs in the human genome, denoted as TPPP1-TPPP3. TPPP1 and TPPP3 are intrinsically unstructured proteins (IUPs) that bind and polymerize tubulin and stabilize microtubules, but TPPP2 does not. Vertebrate TPPPs originated from the ancient invertebrate TPPP by two-round whole-genome duplication; thus, whether the tubulin/microtubule binding function of TPPP1 and TPPP3 is a newly acquired property or was present in the invertebrate orthologs (generally one TPPP per species) has been an open question. To answer this question, we investigated a TPPP from a simple and early branching animal, the sponge Suberites domuncula. Bioinformatics, biochemical, immunochemical, spectroscopic, and electron microscopic data showed that the properties of the sponge protein correspond to those of TPPP1; namely, it is an IUP that strongly binds tubulin and induces its polymerization, proving that these features of animal TPPPs have been evolutionarilyconserved.

S100 fused-type proteins (SFTPs) such as filaggrin, trichohyalin, and cornulin are differentially expressed in cornifying keratinocytes of the epidermis and various skin appendages. To determine evolutionarilyconserved, and thus presumably important, features of SFTPs, we characterized nonmammalian SFTPs and compared their amino acid sequences and expression patterns with those of mammalian SFTPs. We identified an ortholog of cornulin and a previously unknown SFTP, termed scaffoldin, in reptiles and birds, whereas filaggrin was confined to mammals. In contrast to mammalian SFTPs, both cornulin and scaffoldin of the chicken are expressed in the embryonic periderm. However, scaffoldin resembles mammalian trichohyalin with regard to its expression in the filiform papillae of the tongue and in the epithelium underneath the forming tips of the claws. Furthermore, scaffoldin is expressed in the epithelial sheath around growing feathers, reminiscent of trichohyalin expression in the inner root sheath of hair. The results of this study show that SFTP-positive epithelia function as scaffolds for the growth of diverse skin appendages such as claws, nails, hair, and feathers, indicating a common evolutionary origin.

Full Text Available Plants possess a sophisticated immune system to recognize and respond to microbial threats in their environment. The level of immune signaling must be tightly regulated so that immune responses can be quickly activated in the presence of pathogens, while avoiding autoimmunity. HSP90s, along with their diverse array of co-chaperones, forms chaperone complexes that have been shown to play both positive and negative roles in regulating the accumulation of immune receptors and regulators. In this study, we examined the role of AtCHIP, an evolutionarilyconserved E3 ligase that was known to interact with chaperones including HSP90s in multicellular organisms including fruit fly, C. elegans, plants and human. Atchip knockout mutants display enhanced disease susceptibility to a virulent oomycete pathogen, and overexpression of AtCHIP causes enhanced disease resistance at low temperature. Although CHIP was reported to target HSP90 for ubiquitination and degradation, accumulation of HSP90.3 was not affected in Atchip plants. In addition, protein accumulation of nucleotide-binding, leucine-rich repeat domain immune receptor (NLR SNC1 is not altered in Atchip mutant. Thus, while AtCHIP plays a role in immunity, it does not seem to regulate the turnover of HSP90 or SNC1. Further investigation is needed in order to determine the exact mechanism behind AtCHIP’s role in regulating plant immune responses.

Sir2 is a NAD+-dependent protein deacetylase that extends lifespan in yeast and worms. This study examines seven human proteins homologous to Sir2 (SIRT1 through SIRT7) for cellular localization, expression profiles, protein deacetylation activity, and effects on human cell lifespan. We found that: 1) three nuclear SIRT proteins (SIRT1, SIRT6, and SIRT7) show different subnuclear localizations: SIRT6 and SIRT7 are associated with heterochromatic regions and nucleoli, respectively, where yeast Sir2 functions; 2) SIRT3, SIRT4, and SIRT5 are localized in mitochondria, an organelle that links aging and energy metabolism; 3) cellular p53 is a major in vivo substrate of SIRT1 deacetylase, but not the other six SIRT proteins; 4) SIRT1, but not the other two nuclear SIRT proteins, shows an in vitro deacetylase activity on histone H4 and p53 peptides; and 5) overexpression of any one of the seven SIRT proteins does not extend cellular replicative lifespan in normal human fibroblasts or prostate epithelial cells. This study supports the notion that multiple human SIRT proteins have evolutionarilyconserved and nonconserved functions at different cellular locations and reveals that the lifespan of normal human cells, in contrast to that of lower eukaryotes, cannot be manipulated by increased expression of a single SIRT protein.

Slit2 is a neural axon guidance and chemorepellent protein that stimulates motility in a variety of cell types. The role of Slit2 in neural development and neoplastic growth and migration has been well established, while the genetic mechanisms underlying regulation of the Slit2 gene have not. We identified the core and proximal promoter of Slit2 by mapping multiple transcriptional start sites, analyzing transcriptional activity, and confirming sequence homology for the Slit2 proximal promoter among a number of species. Deletion series and transient transfection identified the Slit2 proximal promoter as within 399 base pairs upstream of the start of transcription. A crucial region for full expression of the Slit2 proximal promoter lies between 399 base pairs and 296 base pairs upstream of the start of transcription. Computer modeling identified three transcription factor-binding consensus sites within this region, of which only site-directed mutagenesis of one of the two identified Sp1 consensus sites inhibited transcriptional activity of the Slit2 proximal promoter (-399 to +253). Bioinformatics analysis of the Slit2 proximal promoter -399 base pair to -296 base pair region shows high sequence conservation over twenty-two species, and that this region follows an expected pattern of sequence divergence through evolution.

Full Text Available The eukaryotic Meiotic Recombination protein 11 (Mre11 plays pivotal roles in the DNA damage response (DDR. Specifically, Mre11 senses and signals DNA double strand breaks (DSB and facilitates their repair through effector proteins belonging to either homologous recombination (HR or non-homologous end joining (NHEJ repair mechanisms. In the human malaria parasite Plasmodium falciparum, HR and alternative-NHEJ have been identified; however, little is known about the upstream factors involved in the DDR of this organism. In this report, we identify a putative ortholog of Mre11 in P. falciparum (PfalMre11 that shares 22% sequence similarity to human Mre11. Homology modeling reveals striking structural resemblance of the predicted PfalMre11 nuclease domain to the nuclease domain of Saccharomyces cerevisiae Mre11 (ScMre11. Complementation analyses reveal functional conservation of PfalMre11 nuclease activity as demonstrated by the ability of the PfalMre11 nuclease domain, in conjunction with the C-terminal domain of ScMre11, to functionally complement an mre11 deficient yeast strain. Functional complementation was virtually abrogated by an amino acid substitution in the PfalMre11 nuclease domain (D398N. PfalMre11 is abundant in the mitotically active trophozoite and schizont stages of P. falciparum and is up-regulated in response to DNA damage, suggesting a role in the DDR. PfalMre11 exhibits physical interaction with PfalRad50. In addition, yeast 2-hybrid studies show that PfalMre11 interacts with ScRad50 and ScXrs2, two important components of the well characterized Mre11-Rad50-Xrs2 complex which is involved in DDR signaling and repair in S. cerevisiae, further supporting a role for PfalMre11 in the DDR. Taken together, these findings provide evidence that PfalMre11 is an evolutionarilyconserved component of the DDR in Plasmodium.

The eukaryotic Meiotic Recombination protein 11 (Mre11) plays pivotal roles in the DNA damage response (DDR). Specifically, Mre11 senses and signals DNA double strand breaks (DSB) and facilitates their repair through effector proteins belonging to either homologous recombination (HR) or non-homologous end joining (NHEJ) repair mechanisms. In the human malaria parasite Plasmodium falciparum, HR and alternative-NHEJ have been identified; however, little is known about the upstream factors involved in the DDR of this organism. In this report, we identify a putative ortholog of Mre11 in P. falciparum (PfalMre11) that shares 22% sequence similarity to human Mre11. Homology modeling reveals striking structural resemblance of the predicted PfalMre11 nuclease domain to the nuclease domain of Saccharomyces cerevisiae Mre11 (ScMre11). Complementation analyses reveal functional conservation of PfalMre11 nuclease activity as demonstrated by the ability of the PfalMre11 nuclease domain, in conjunction with the C-terminal domain of ScMre11, to functionally complement an mre11 deficient yeast strain. Functional complementation was virtually abrogated by an amino acid substitution in the PfalMre11 nuclease domain (D398N). PfalMre11 is abundant in the mitotically active trophozoite and schizont stages of P. falciparum and is up-regulated in response to DNA damage, suggesting a role in the DDR. PfalMre11 exhibits physical interaction with PfalRad50. In addition, yeast 2-hybrid studies show that PfalMre11 interacts with ScRad50 and ScXrs2, two important components of the well characterized Mre11-Rad50-Xrs2 complex which is involved in DDR signaling and repair in S. cerevisiae, further supporting a role for PfalMre11 in the DDR. Taken together, these findings provide evidence that PfalMre11 is an evolutionarilyconserved component of the DDR in Plasmodium.

Tight junctions are the structures in mammalian epithelial cells that separate the apical and basolateral membranes and may also be important in the establishment of cell polarity. Two evolutionarilyconserved multiprotein complexes, Crumbs-PALS1 (Stardust)-PATJ and Cdc42-Par6-Par3-atypical protein kinase C, have been implicated in the assembly of tight junctions and in polarization of Drosophila melanogaster epithelia. These two complexes have been linked physically and functionally by an interaction between PALS1 and Par6. Here we identify an evolutionarilyconserved region in the amino terminus of PALS1 as the Par6 binding site and identify valine and aspartic acid residues in this region as essential for interacting with the PDZ domain of Par6. We have also characterized, in more detail, the amino terminus of Drosophila Stardust and demonstrate that the interaction mechanism between Stardust and Drosophila Par6 is evolutionarilyconserved. Par6 interferes with PATJ in binding PALS1, and these two interactions do not appear to function synergistically. Taken together, these results define the molecular mechanisms linking two conserved polarity complexes.

The intestinal epithelium serves critical physiologic functions that are shared among all vertebrates. However, it is unknown how the transcriptional regulatory mechanisms underlying these functions have changed over the course of vertebrate evolution. We generated genome-wide mRNA and accessible chromatin data from adult intestinal epithelial cells (IECs) in zebrafish, stickleback, mouse, and human species to determine if conserved IEC functions are achieved through common transcriptional regulation. We found evidence for substantial common regulation and conservation of gene expression regionally along the length of the intestine from fish to mammals and identified a core set of genes comprising a vertebrate IEC signature. We also identified transcriptional start sites and other putative regulatory regions that are differentially accessible in IECs in all 4 species. Although these sites rarely showed sequence conservation from fish to mammals, surprisingly, they drove highly conserved IEC expression in a zebrafish reporter assay. Common putative transcription factor binding sites (TFBS) found at these sites in multiple species indicate that sequence conservation alone is insufficient to identify much of the functionally conserved IEC regulatory information. Among the rare, highly sequence-conserved, IEC-specific regulatory regions, we discovered an ancient enhancer upstream from her6/HES1 that is active in a distinct population of Notch-positive cells in the intestinal epithelium. Together, these results show how combining accessible chromatin and mRNA datasets with TFBS prediction and in vivo reporter assays can reveal tissue-specific regulatory information conserved across 420 million years of vertebrate evolution. We define an IEC transcriptional regulatory network that is shared between fish and mammals and establish an experimental platform for studying how evolutionarily distilled regulatory information commonly controls IEC development and physiology. PMID

SoxB transcription factors and histone deacetylases (HDACs) are each major players in the regulation of neurogenesis, but a functional link between them has not been previously demonstrated. Here, we show that SoxB2 and Hdac2 act together to regulate neurogenesis in the cnidarian Hydractinia echinata during tissue homeostasis and head regeneration. We find that misexpression of SoxB genes modifies the number of neural cells in all life stages and interferes with head regeneration. Hdac2 was co-expressed with SoxB2, and its downregulation phenocopied SoxB2 knockdown. We also show that SoxB2 and Hdac2 promote each other's transcript levels, but Hdac2 counteracts this amplification cycle by deacetylating and destabilizing SoxB2 protein. Finally, we present evidence for conservation of these interactions in human neural progenitors. We hypothesize that crosstalk between SoxB transcription factors and Hdac2 is an ancient feature of metazoan neurogenesis and functions to stabilize the correct levels of these multifunctional proteins.

Full Text Available To elucidate the transcriptional regulation of Bmp4 expression during organogenesis, we used phylogenetic footprinting and transgenic reporter analyses to identify Bmp4 cis-regulatory modules (CRMs. These analyses identified a regulatory region located ∼46 kb upstream of the mouse Bmp4 transcription start site that had previously been shown to direct expression in lateral plate mesoderm. We refined this regulatory region to a 396-bp minimal enhancer, and show that it recapitulates features of endogenous Bmp4 expression in developing mandibular arch ectoderm and incisor epithelium during the initiation-stage of tooth development. In addition, this enhancer directs expression in the apical ectodermal ridge (AER of the developing limb and in anterior and posterior limb mesenchyme. Transcript profiling of E11.5 mouse incisor dental lamina, together with protein binding microarray (PBM analyses, allowed identification of a conserved DNA binding motif in the Bmp4 enhancer for Pitx homeoproteins, which are also expressed in the developing mandibular and incisor epithelium. In vitro electrophoretic mobility shift assays (EMSA and in vivo transgenic reporter mutational analyses revealed that this site supports Pitx binding and that the site is necessary to recapitulate aspects of endogenous Bmp4 expression in developing craniofacial and limb tissues. Finally, Pitx2 chromatin immunoprecipitation (ChIP demonstrated direct binding of Pitx2 to this Bmp4 enhancer site in a dental epithelial cell line. These results establish a direct molecular regulatory link between Pitx family members and Bmp4 gene expression in developing incisor epithelium.

The mechanisms that tightly control the transcription of host defense genes have not been fully elucidated. We previously identified TFEB as a transcription factor important for host defense, but the mechanisms that regulate TFEB during infection remained unknown. Here, we used C. elegans to discover a pathway that activates TFEB during infection. Gene dkf-1, which encodes a homolog of protein kinase D (PKD), was required for TFEB activation in nematodes infected with Staphylococcus aureus. Conversely, pharmacological activation of PKD was sufficient to activate TFEB. Furthermore, phospholipase C (PLC) gene plc-1 was also required for TFEB activation, downstream of Gαq homolog egl-30 and upstream of dkf-1. Using reverse and chemical genetics, we discovered a similar PLC-PKD-TFEB axis in Salmonella-infected mouse macrophages. In addition, PKCα was required in macrophages. These observations reveal a previously unknown host defense signaling pathway, which has been conserved across one billion years of evolution.

Full Text Available Abstract Background Gcn5 is a transcriptional coactivator with histone acetyltransferase activity that is conserved with regard to structure as well as its histone substrates throughout the eukaryotes. Gene regulatory networks within cells are thought to be evolutionarily diverged. The use of evolutionarily divergent yeast species, such as S. cerevisiae and S. pombe, which can be studied under similar environmental conditions, provides an opportunity to examine the interface between conserved regulatory components and their cellular applications in different organisms. Results We show that Gcn5 is important for a common set of stress responses in evolutionarily diverged yeast species and that the activity of the conserved histone acetyltransferase domain is required. We define a group of KCl stress response genes in S. cerevisiae that are specifically dependent on Gcn5. Gcn5 is localised to many Gcn5-dependent genes including Gcn5 repressed targets such as FLO8. Gcn5 regulates divergent sets of KCl responsive genes in S. cerevisiae and S. pombe. Genome-wide localization studies showed a tendency for redistribution of Gcn5 during KCl stress adaptation in S. cerevisiae from short genes to the transcribed regions of long genes. An analogous redistribution was not observed in S. pombe. Conclusions Gcn5 is required for the regulation of divergent sets of KCl stress-response genes in S. cerevisiae and S. pombe even though it is required a common group of stress responses, including the response to KCl. Genes that are physically associated with Gcn5 require its activity for their repression or activation during stress adaptation, providing support for a role of Gcn5 as a corepressor as well as a coactivator. The tendency of Gcn5 to re-localise to the transcribed regions of long genes during KCl stress adaptation suggests that Gcn5 plays a specific role in the expression of long genes under adaptive conditions, perhaps by regulating transcriptional

Both the Prp18 protein and the U5 snRNA function in the second step of pre-mRNA splicing. We identified suppressors of mutant prp18 alleles in the gene for the U5 snRNA (SNR7). The suppressors' U5 snRNAs have either a U4-to-A or an A8-to-C mutation in the evolutionarily invariant loop 1 of U5. Suppression is specific for prp18 alleles that encode proteins with mutations in a highly conserved region of Prp18 which forms an unstructured loop in crystals of Prp18. The snr7 suppressors partly res...

Dietary restriction (DR), limiting nutrient intake from diet without causing malnutrition, delays the aging process and extends lifespan in multiple organisms. The conserved life-extending effect of DR suggests the involvement of fundamental mechanisms, although these remain a subject of debate. To help decipher the life-extending mechanisms of DR, we first compiled a list of genes that if genetically altered disrupt or prevent the life-extending effects of DR. We called these DR-essential genes and identified more than 100 in model organisms such as yeast, worms, flies, and mice. In order for other researchers to benefit from this first curated list of genes essential for DR, we established an online database called GenDR (http://genomics.senescence.info/diet/). To dissect the interactions of DR-essential genes and discover the underlying lifespan-extending mechanisms, we then used a variety of network and systems biology approaches to analyze the gene network of DR. We show that DR-essential genes are more conserved at the molecular level and have more molecular interactions than expected by chance. Furthermore, we employed a guilt-by-association method to predict novel DR-essential genes. In budding yeast, we predicted nine genes related to vacuolar functions; we show experimentally that mutations deleting eight of those genes prevent the life-extending effects of DR. Three of these mutants (OPT2, FRE6, and RCR2) had extended lifespan under ad libitum, indicating that the lack of further longevity under DR is not caused by a general compromise of fitness. These results demonstrate how network analyses of DR using GenDR can be used to make phenotypically relevant predictions. Moreover, gene-regulatory circuits reveal that the DR-induced transcriptional signature in yeast involves nutrient-sensing, stress responses and meiotic transcription factors. Finally, comparing the influence of gene expression changes during DR on the interactomes of multiple organisms led

Full Text Available Dietary restriction (DR, limiting nutrient intake from diet without causing malnutrition, delays the aging process and extends lifespan in multiple organisms. The conserved life-extending effect of DR suggests the involvement of fundamental mechanisms, although these remain a subject of debate. To help decipher the life-extending mechanisms of DR, we first compiled a list of genes that if genetically altered disrupt or prevent the life-extending effects of DR. We called these DR-essential genes and identified more than 100 in model organisms such as yeast, worms, flies, and mice. In order for other researchers to benefit from this first curated list of genes essential for DR, we established an online database called GenDR (http://genomics.senescence.info/diet/. To dissect the interactions of DR-essential genes and discover the underlying lifespan-extending mechanisms, we then used a variety of network and systems biology approaches to analyze the gene network of DR. We show that DR-essential genes are more conserved at the molecular level and have more molecular interactions than expected by chance. Furthermore, we employed a guilt-by-association method to predict novel DR-essential genes. In budding yeast, we predicted nine genes related to vacuolar functions; we show experimentally that mutations deleting eight of those genes prevent the life-extending effects of DR. Three of these mutants (OPT2, FRE6, and RCR2 had extended lifespan under ad libitum, indicating that the lack of further longevity under DR is not caused by a general compromise of fitness. These results demonstrate how network analyses of DR using GenDR can be used to make phenotypically relevant predictions. Moreover, gene-regulatory circuits reveal that the DR-induced transcriptional signature in yeast involves nutrient-sensing, stress responses and meiotic transcription factors. Finally, comparing the influence of gene expression changes during DR on the interactomes of

Dietary restriction (DR), limiting nutrient intake from diet without causing malnutrition, delays the aging process and extends lifespan in multiple organisms. The conserved life-extending effect of DR suggests the involvement of fundamental mechanisms, although these remain a subject of debate. To help decipher the life-extending mechanisms of DR, we first compiled a list of genes that if genetically altered disrupt or prevent the life-extending effects of DR. We called these DR–essential genes and identified more than 100 in model organisms such as yeast, worms, flies, and mice. In order for other researchers to benefit from this first curated list of genes essential for DR, we established an online database called GenDR (http://genomics.senescence.info/diet/). To dissect the interactions of DR–essential genes and discover the underlying lifespan-extending mechanisms, we then used a variety of network and systems biology approaches to analyze the gene network of DR. We show that DR–essential genes are more conserved at the molecular level and have more molecular interactions than expected by chance. Furthermore, we employed a guilt-by-association method to predict novel DR–essential genes. In budding yeast, we predicted nine genes related to vacuolar functions; we show experimentally that mutations deleting eight of those genes prevent the life-extending effects of DR. Three of these mutants (OPT2, FRE6, and RCR2) had extended lifespan under ad libitum, indicating that the lack of further longevity under DR is not caused by a general compromise of fitness. These results demonstrate how network analyses of DR using GenDR can be used to make phenotypically relevant predictions. Moreover, gene-regulatory circuits reveal that the DR–induced transcriptional signature in yeast involves nutrient-sensing, stress responses and meiotic transcription factors. Finally, comparing the influence of gene expression changes during DR on the interactomes of multiple

The expression of specific transcription factors determines the differentiated features of postmitotic neurons. However, the mechanism by which specific molecules determine neuronal cell fate and the extent to which the functions of transcription factors are conserved in evolution are not fully understood. In C. elegans, the cholinergic and peptidergic SMB sensory/inter/motor neurons innervate muscle quadrants in the head and control the amplitude of sinusoidal movement. Here we show that the LIM homeobox protein LIM-4 determines neuronal characteristics of the SMB neurons. In lim-4 mutant animals, expression of terminal differentiation genes, such as the cholinergic gene battery and the flp-12 neuropeptide gene, is completely abolished and thus the function of the SMB neurons is compromised. LIM-4 activity promotes SMB identity by directly regulating the expression of the SMB marker genes via a distinct cis-regulatory motif. Two human LIM-4 orthologs, LHX6 and LHX8, functionally substitute for LIM-4 in C. elegans. Furthermore, C. elegans LIM-4 or human LHX6 can induce cholinergic and peptidergic characteristics in the human neuronal cell lines. Our results indicate that the evolutionarilyconserved LIM-4/LHX6 homeodomain proteins function in generation of precise neuronal subtypes. PMID:26305787

Full Text Available Immature dendritic cells (DCs maintain a highly dynamic pool of recycling MHCII that promotes sampling of environmental antigens for presentation to T helper cells. However, the molecular basis of MHCII recycling and the cellular machinery that orchestrates MHCII trafficking are incompletely understood. Using a mouse model we show that WASH, an actin regulatory protein that facilitates retromer function, is essential for MHCII recycling and efficient priming of T helper cells. We further demonstrate that WASH deficiency results in impaired MHCII surface levels, recycling, and an accumulation of polyubiquitinated MHCII complexes, which are subsequently slated for premature lysosomal degradation. Consequently, conditional deletion of the Wash gene in DCs impairs priming of both conventional and autoimmune T helper cells in vivo and attenuates disease progression in a model of experimental autoimmune encephalitis (EAE. Thus, we identify a novel mechanism in which DCs employ the evolutionarilyconserved WASH and retromer complex for MHCII recycling in order to regulate T helper cell priming.

Full Text Available Overexpression of human epidermal growth factor receptor 2 (HER2 is associated with tumor aggressiveness and poor prognosis in breast cancer. With the availability of therapeutic antibodies against HER2, great strides have been made in the clinical management of HER2 overexpressing breast cancer. However, de novo and acquired resistance to these antibodies presents a serious limitation to successful HER2 targeting treatment. The identification of novel epitopes of HER2 that can be used for functional/region-specific blockade could represent a central step in the development of new clinically relevant anti-HER2 antibodies. In the present study, we present a novel computational approach as an auxiliary tool for identification of novel HER2 epitopes. We hypothesized that the structurally and linearly evolutionarilyconserved motifs of the extracellular domain of HER2 (ECD HER2 contain potential druggable epitopes/targets. We employed the PROSITE Scan to detect structurally conserved motifs and PRINTS to search for linearly conserved motifs of ECD HER2. We found that the epitopes recognized by trastuzumab and pertuzumab are located in the predicted conserved motifs of ECD HER2, supporting our initial hypothesis. Considering that structurally and linearly conserved motifs can provide functional specific configurations, we propose that by comparing the two types of conserved motifs, additional druggable epitopes/targets in the ECD HER2 protein can be identified, which can be further modified for potential therapeutic application. Thus, this novel computational process for predicting or searching for potential epitopes or key target sites may contribute to epitope-based vaccine and function-selected drug design, especially when x-ray crystal structure protein data is not available.

Full Text Available Alternative splicing (AS plays important roles in many plant functions, but its conservation across the plant kingdom is not known. We describe a methodology to identify AS events and identify conserved AS events across large phylogenetic distances using RNA-Seq datasets. We applied this methodology to transcriptome data from nine angiosperms including Amborella, the single sister species to all other extant flowering plants. AS events within 40 – 70% of the expressed multi-exonic genes per species were found, 27,120 of which are conserved among two or more of the taxa studied. While many events are species specific, many others are shared across long evolutionary distances suggesting they have functional significance. Conservation of AS event data provides an estimate of the number of ancestral AS events present at each node of the tree representing the 9 species studied. Furthermore, the presence or absence of AS isoforms between species with different whole genome duplication (WGD histories provides the opportunity to examine the impact of WDG on AS potential. Examining AS in gene families identifies those with high rates of AS, and conservation can distinguish ancient events vs. recent or species specific adaptations. The MADS-box and SR protein families are found to represent families with low and high occurances of AS, respectively, yet their AS events were likely present in the MRCA of angiosperms.

Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of gi...

Ultraconserved elements, unusually long regions of perfect sequence identity, are found in genes encoding numerous RNA-binding proteins including arginine-serine rich (SR) splicing factors. Expression of these genes is regulated via alternative splicing of the ultraconserved regions to yield mRNAs that are degraded by nonsense-mediated mRNA decay (NMD), a process termed unproductive splicing (Lareau et al. 2007; Ni et al. 2007). As all human SR genes are affected by alternative splicing and NMD, one might expect this regulation to have originated in an early SR gene and persisted as duplications expanded the SR family. But in fact, unproductive splicing of most human SR genes arose independently (Lareau et al. 2007). This paradox led us to investigate the origin and proliferation of unproductive splicing in SR genes. We demonstrate that unproductive splicing of the splicing factor SRSF5 (SRp40) is conserved among all animals and even observed in fungi; this is a rare example of alternative splicing conserved between kingdoms, yet its effect is to trigger mRNA degradation. As the gene duplicated, the ancient unproductive splicing was lost in paralogs, and distinct unproductive splicing evolved rapidly and repeatedly to take its place. SR genes have consistently employed unproductive splicing, and while it is exceptionally conserved in some of these genes, turnover in specific events among paralogs shows flexible means to the same regulatory end.

Full Text Available Alternative splicing amplifies the information content of the genome, creating multiple mRNA isoforms from single genes. The evolutionarilyconserved splicing activator Tra2β (Sfrs10 is essential for mouse embryogenesis and implicated in spermatogenesis. Here we find that Tra2β is up-regulated as the mitotic stem cell containing population of male germ cells differentiate into meiotic and post-meiotic cells. Using CLIP coupled to deep sequencing, we found that Tra2β binds a high frequency of exons and identified specific G/A rich motifs as frequent targets. Significantly, for the first time we have analysed the splicing effect of Sfrs10 depletion in vivo by generating a conditional neuronal-specific Sfrs10 knock-out mouse (Sfrs10(fl/fl; Nestin-Cre(tg/+. This mouse has defects in brain development and allowed correlation of genuine physiologically Tra2β regulated exons. These belonged to a novel class which were longer than average size and importantly needed multiple cooperative Tra2β binding sites for efficient splicing activation, thus explaining the observed splicing defects in the knockout mice. Regulated exons included a cassette exon which produces a meiotic isoform of the Nasp histone chaperone that helps monitor DNA double-strand breaks. We also found a previously uncharacterised poison exon identifying a new pathway of feedback control between vertebrate Tra2 proteins. Both Nasp-T and the Tra2a poison exon are evolutionarilyconserved, suggesting they might control fundamental developmental processes. Tra2β protein isoforms lacking the RRM were able to activate specific target exons indicating an additional functional role as a splicing co-activator. Significantly the N-terminal RS1 domain conserved between flies and humans was essential for the splicing activator function of Tra2β. Versions of Tra2β lacking this N-terminal RS1 domain potently repressed the same target exons activated by full-length Tra2β protein.

Both the Prp18 protein and the U5 snRNA function in the second step of pre-mRNA splicing. We identified suppressors of mutant prp18 alleles in the gene for the U5 snRNA (SNR7). The suppressors' U5 snRNAs have either a U4-to-A or an A8-to-C mutation in the evolutionarily invariant loop 1 of U5. Suppression is specific for prp18 alleles that encode proteins with mutations in a highly conserved region of Prp18 which forms an unstructured loop in crystals of Prp18. The snr7 suppressors partly restored the pre-mRNA splicing activity that was lost in the prp18 mutants. The close functional relationship of Prp18 and U5 is emphasized by the finding that two snr7 alleles, U5A and U6A, are dominant synthetic lethal with prp18 alleles. Our results support the idea that Prp18 and the U5 snRNA act in concert during the second step of pre-mRNA splicing and suggest a model in which the conserved loop of Prp18 acts to stabilize the interaction of loop 1 of the U5 snRNA with the splicing intermediates.

Full Text Available Parasites of the Leishmania genus can rapidly alter several macrophage (MØ signalling pathways in order to tame down the innate immune response and inflammation, therefore favouring their survival and propagation within their mammalian host. Having recently reported that Leishmania and bacterial LPS generate a significantly stronger inflammatory response in animals and phagocytes functionally deficient for the Src homology 2 domain-containing protein tyrosine phosphatase (SHP-1, we hypothesized that Leishmania could exploit SHP-1 to inactivate key kinases involved in Toll-like receptor (TLR signalling and innate immunity such as IL-1 receptor-associated kinase 1 (IRAK-1. Here we show that upon infection, SHP-1 rapidly binds to IRAK-1, completely inactivating its intrinsic kinase activity and any further LPS-mediated activation as well as MØ functions. We also demonstrate that the SHP-1/IRAK-1 interaction occurs via an evolutionarilyconserved ITIM-like motif found in the kinase domain of IRAK-1, which we named KTIM (Kinase Tyrosyl-based Inhibitory Motif. This regulatory motif appeared in early vertebrates and is not found in any other IRAK family member. Our study additionally reveals that several other kinases (e.g. Erk1/2, IKKalpha/beta involved in downstream TLR signalling also bear KTIMs in their kinase domains and interact with SHP-1. We thus provide the first demonstration that a pathogen can exploit a host protein tyrosine phosphatase, namely SHP-1, to directly inactivate IRAK-1 through a generally conserved KTIM motif.

In eukaryotes, it is generally assumed that translation initiation occurs at the AUG codon closest to the messenger RNA 5\\' cap. However, in certain cases, initiation can occur at codons differing from AUG by a single nucleotide, especially the codons CUG, UUG, GUG, ACG, AUA and AUU. While non-AUG initiation has been experimentally verified for a handful of human genes, the full extent to which this phenomenon is utilized--both for increased coding capacity and potentially also for novel regulatory mechanisms--remains unclear. To address this issue, and hence to improve the quality of existing coding sequence annotations, we developed a methodology based on phylogenetic analysis of predicted 5\\' untranslated regions from orthologous genes. We use evolutionary signatures of protein-coding sequences as an indicator of translation initiation upstream of annotated coding sequences. Our search identified novel conserved potential non-AUG-initiated N-terminal extensions in 42 human genes including VANGL2, FGFR1, KCNN4, TRPV6, HDGF, CITED2, EIF4G3 and NTF3, and also affirmed the conservation of known non-AUG-initiated extensions in 17 other genes. In several instances, we have been able to obtain independent experimental evidence of the expression of non-AUG-initiated products from the previously published literature and ribosome profiling data.

The enzyme Dicer generates 21-25 nucleotide RNAs that target specific mRNAs for silencing during RNA interference and related pathways. Although their active sites and RNA binding regions are functionally conserved, the helicase domains have distinct activities in the context of different Dicer enzymes. To examine the evolutionary origins of Dicer helicase functions, we investigated two related Dicer enzymes from the thermophilic fungus Sporotrichum thermophile. RNA cleavage assays showed that S. thermophile Dicer-1 (StDicer-1) can process hairpin precursor microRNAs, whereas StDicer-2 can only cleave linear double-stranded RNAs. Furthermore, only StDicer-2 possesses robust ATP hydrolytic activity in the presence of double-stranded RNA. Deletion of the StDicer-2 helicase domain increases both StDicer-2 cleavage activity and affinity for hairpin RNA. Notably, both StDicer-1 and StDicer-2 could complement the distantly related yeast Schizosaccharomyces pombe lacking its endogenous Dicer gene but only in their full-length forms, underscoring the importance of the helicase domain. These results suggest an in vivo regulatory function for the helicase domain that may be conserved from fungi to humans.

Full Text Available Cockayne syndrome (CS is a devastating progeria most often caused by mutations in the CSB gene encoding a SWI/SNF family chromatin remodeling protein. Although all CSB mutations that cause CS are recessive, the complete absence of CSB protein does not cause CS. In addition, most CSB mutations are located beyond exon 5 and are thought to generate only C-terminally truncated protein fragments. We now show that a domesticated PiggyBac-like transposon PGBD3, residing within intron 5 of the CSB gene, functions as an alternative 3' terminal exon. The alternatively spliced mRNA encodes a novel chimeric protein in which CSB exons 1-5 are joined in frame to the PiggyBac transposase. The resulting CSB-transposase fusion protein is as abundant as CSB protein itself in a variety of human cell lines, and continues to be expressed by primary CS cells in which functional CSB is lost due to mutations beyond exon 5. The CSB-transposase fusion protein has been highly conserved for at least 43 Myr since the divergence of humans and marmoset, and appears to be subject to selective pressure. The human genome contains over 600 nonautonomous PGBD3-related MER85 elements that were dispersed when the PGBD3 transposase was last active at least 37 Mya. Many of these MER85 elements are associated with genes which are involved in neuronal development, and are known to be regulated by CSB. We speculate that the CSB-transposase fusion protein has been conserved for host antitransposon defense, or to modulate gene regulation by MER85 elements, but may cause CS in the absence of functional CSB protein.

Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of giActin produced filaments, indicating that this divergent actin is a true filament-forming actin. We generated an anti-giActin antibody to localize giActin throughout the cell cycle. GiActin localized to the cortex, nuclei, internal axonemes, and formed C-shaped filaments along the anterior of the cell and a flagella-bundling helix. These structures were regulated with the cell cycle and in encysting cells giActin was recruited to the Golgi-like cyst wall processing vesicles. Knockdown of giActin demonstrated that giActin functions in cell morphogenesis, membrane trafficking, and cytokinesis. Additionally, Giardia contains a single G protein, giRac, which affects the Giardia actin cytoskeleton independently of known target ABPs. These results imply that there exist ancestral and perhaps conserved roles for actin in core cellular processes that are independent of canonical ABPs. Of medical significance, the divergent giActin cytoskeleton is essential and commonly used actin-disrupting drugs do not depolymerize giActin structures. Therefore, the giActin cytoskeleton is a promising drug target for treating giardiasis, as we predict drugs that interfere with the Giardia actin cytoskeleton will not affect the mammalian host.

A novel exon microarray format that probes gene expression with single exon resolution was employed to elucidate critical features of a vertebrate muscle alternative splicing program. A dataset of 56 microarray-defined, muscle-enriched exons and their flanking introns were examined computationally in order to investigate coordination of the muscle splicing program. Candidate intron regulatory motifs were required to meet several stringent criteria: significant over-representation near muscle-enriched exons, correlation with muscle expression, and phylogenetic conservation among genomes of several vertebrate orders. Three classes of regulatory motifs were identified in the proximal downstream intron, within 200nt of the target exons: UGCAUG, a specific binding site for Fox-1 related splicing factors; ACUAAC, a novel branchpoint-like element; and UG-/UGC-rich elements characteristic of binding sites for CELF splicing factors. UGCAUG was remarkably enriched, being present in nearly one-half of all cases. These studies suggest that Fox and CELF splicing factors play a major role in enforcing the muscle-specific alternative splicing program, facilitating expression of a set of unique isoforms of cytoskeletal proteins that are critical to muscle cell differentiation. Supplementary materials: There are four supplementary tables and one supplementary figure. The tables provide additional detailed information concerning the muscle-enriched datasets, and about over-represented oligonucleotide sequences in the flanking introns. The supplementary figure shows RT-PCR data confirming the muscle-enriched expression of exons predicted from the microarray analysis.

The bacterial hibernating 100S ribosome is a poorly understood form of the dimeric 70S particle that has been linked to pathogenesis, translational repression, starvation responses, and ribosome turnover. In the opportunistic pathogen Staphylococcus aureus and most other bacteria, hibernation-promoting factor (HPF) homodimerizes the 70S ribosomes to form a translationally silent 100S complex. Conversely, the 100S ribosomes dissociate into subunits and are presumably recycled for new rounds of translation. The regulation and disassembly of the 100S ribosome are largely unknown because the temporal abundance of the 100S ribosome varies considerably among different bacterial phyla. Here, we identify a universally conserved GTPase (HflX) as a bona fide dissociation factor of the S. aureus 100S ribosome. The expression levels hpf and hflX are coregulated by general stress and stringent responses in a temperature-dependent manner. While all tested guanosine analogs stimulate the splitting activity of HflX on the 70S ribosome, only GTP can completely dissociate the 100S ribosome. Our results reveal the antagonistic relationship of HPF and HflX and uncover the key regulators of 70S and 100S ribosome homeostasis that are intimately associated with bacterial survival.

The immune system is based on the actions of the collection of specialized immune defense cells and their secreted proteins and peptides that defend the host against infection by parasites. Parasites are organisms that live part or all of their lives in close physical association with the host and extract nutrients from the host and, by releasing toxins and virulence factors, cause disease with the potential for injury and premature death of that host. Parasites of the metazoa can be viruses, eubacteria, fungi, protozoans, and other metazoans. The immune system operates to kill or eliminate parasites and eliminate or detoxify their toxins and virulence factors. Although some of the elements of immune systems are specific to a particular phylum of metazoans, others show extensive evolutionary conservation, being present in several or all major phyla of the metazoa. The pentraxins display this latter character in their roles in immune defense. Pentraxins have been documented in vertebrates, nonvertebrate chordates, arthropods, and mollusks and may be present in other taxa of metazoans. Presumably the pentraxins appeared early in the evolution of metazoa, prior to their evolutionary divergence in the Precambrian epoch into many phyla present today, and have been preserved for the 542 million years since that explosive evolutionary radiation. The fidelity with which these phyla have preserved the pentraxins suggests that the functions of these proteins are important for survival of the members of these diverse taxa of animals.

By using natural abundance (13)C NMR spectroscopy, we investigated the types of compatible solutes synthesized in a variety of Bacilli under high salinity growth conditions. Glutamate, proline, and ectoine were the dominant compatible solutes synthesized by the various Bacillus species. The majority of the inspected Bacilli produced the tetrahydropyrimidine ectoine in response to high salinity stress, and a subset of these also synthesized a hydroxylation derivative of ectoine, 5-hydroxyectoine. In Salibacillus salexigens, a representative of the ectoine- and 5-hydroxyectoine-producing species, ectoine production was linearly correlated with the salinity of the growth medium and dependent on an ectABC biosynthetic operon. The formation of 5-hydroxyectoine was primarily a stationary growth phase phenomenon. The enzyme responsible for ectoine hydroxylation (EctD) was purified from S. salexigens to apparent homogeneity. The EctD protein was shown in vitro to directly hydroxylate ectoine in a reaction dependent on iron(II), molecular oxygen, and 2-oxoglutarate. We identified the structural gene (ectD) for the ectoine hydroxylase in S. salexigens. Northern blot analysis showed that the transcript levels of the ectABC and ectD genes increased as a function of salinity. Many EctD-related proteins can be found in data base searches in various Bacteria. Each of these bacterial species also contains an ectABC ectoine biosynthetic gene cluster, suggesting that 5-hydroxyectoine biosynthesis strictly depends on the prior synthesis of ectoine. Our data base searches and the biochemical characterization of the EctD protein from S. salexigens suggest that the EctD-related ectoine hydroxylases are members of a new subfamily within the non-heme-containing, iron(II)- and 2-oxoglutarate-dependent dioxygenase superfamily (EC 1.14.11).

Full Text Available Of the five human KCNQ (Kv7 channels, KCNQ1 with auxiliary subunit KCNE1 mediates the native cardiac I(Ks current with mutations causing short and long QT cardiac arrhythmias. KCNQ4 mutations cause deafness. KCNQ2/3 channels form the native M-current controlling excitability of most neurons, with mutations causing benign neonatal febrile convulsions. Drosophila contains a single KCNQ (dKCNQ that appears to serve alone the functions of all the duplicated mammalian neuronal and cardiac KCNQ channels sharing roughly 50-60% amino acid identity therefore offering a route to investigate these channels. Current information about the functional properties of dKCNQ is lacking therefore we have investigated these properties here. Using whole cell patch clamp electrophysiology we compare the biophysical and pharmacological properties of dKCNQ with the mammalian neuronal and cardiac KCNQ channels expressed in HEK cells. We show that Drosophila KCNQ (dKCNQ is a slowly activating and slowly-deactivating K(+ current open at sub-threshold potentials that has similar properties to neuronal KCNQ2/3 with some features of the cardiac KCNQ1/KCNE1 accompanied by conserved sensitivity to a number of clinically relevant KCNQ blockers (chromanol 293B, XE991, linopirdine and opener (zinc pyrithione. We also investigate the molecular basis of the differential selectivity of KCNQ channels to the opener retigabine and show a single amino acid substitution (M217W can confer sensitivity to dKCNQ. We show dKCNQ has similar electrophysiological and pharmacological properties as the mammalian KCNQ channels, allowing future study of physiological and pathological roles of KCNQ in Drosophila and whole organism screening for new modulators of KCNQ channelopathies.

Deacetylases of the Sir2 or sirtuin family are thought to regulate life cycle progression and life span in response to nutrient availability. This family has undergone successive rounds of duplication and diversification, enabling the enzymes to perform a wide variety of biological functions. Two evolutionarilyconserved functions of yeast Sir2 proteins are the generation of repressive chromatin in subtelomeric domains and the suppression of unbalanced recombination within the tandem rDNA array. Here, we describe the function of the Sir2 ortholog ClHst1 in the yeast Clavispora lusitaniae, an occasional opportunistic pathogen. ClHst1 was localized to the non-transcribed spacer regions of the rDNA repeats and deacetylated histones at these loci, indicating that, like other Sir2 proteins, ClHst1 modulates chromatin structure at the rDNA repeats. However, we found no evidence that ClHst1 associates with subtelomeric regions or impacts gene expression directly. This surprising observation highlights the plasticity of sirtuin function. Related yeast species, including Candida albicans, possess an additional Sir2 family member. Thus, it is likely that the ancestral Candida SIR2/HST1 gene was duplicated and subfunctionalized, such that HST1 retained the capacity to regulate rDNA whereas SIR2 had other functions, perhaps including the generation of subtelomeric chromatin. After subsequent species diversification, the SIR2 paralog was apparently lost in the C. lusitaniae lineage. Thus, C. lusitaniae presents an opportunity to discover how subtelomeric chromatin can be reconfigured.

The vertebrate head–trunk interface (occipital region) has been heavily remodelled during evolution, and its development is still poorly understood. In extant jawed vertebrates, this region provides muscle precursors for the throat and tongue (hypopharyngeal/hypobranchial/hypoglossal muscle precursors, HMP) that take a stereotype path rostrally along the pharynx and are thought to reach their target sites via active migration. Yet, this projection pattern emerged in jawless vertebrates before the evolution of migratory muscle precursors. This suggests that a so far elusive, more basic transport mechanism must have existed and may still be traceable today. Here we show for the first time that all occipital tissues participate in well-conserved cell movements. These cell movements are spearheaded by the occipital lateral mesoderm and ectoderm that split into two streams. The rostrally directed stream projects along the floor of the pharynx and reaches as far rostrally as the floor of the mandibular arch and outflow tract of the heart. Notably, this stream leads and engulfs the later emerging HMP, neural crest cells and hypoglossal nerve. When we (i) attempted to redirect hypobranchial/hypoglossal muscle precursors towards various attractants, (ii) placed non-migratory muscle precursors into the occipital environment or (iii) molecularly or (iv) genetically rendered muscle precursors non-migratory, they still followed the trajectory set by the occipital lateral mesoderm and ectoderm. Thus, we have discovered evolutionarilyconserved morphogenetic movements, driven by the occipital lateral mesoderm and ectoderm, that ensure cell transport and organ assembly at the head–trunk interface. PMID:24662046

Gene-expression analysis studies from Schultz et al. estimate that more than 2,300 genes in the mouse genome are expressed predominantly in the male germ line. As of their 2003 publication [Schultz N, Hamra FK, Garbers DL (2003) Proc Natl Acad Sci USA 100(21):12201–12206], the functions of the majority of these testis-enriched genes during spermatogenesis and fertilization were largely unknown. Since the study by Schultz et al., functional analysis of hundreds of reproductive-tract–enriched genes have been performed, but there remain many testis-enriched genes for which their relevance to reproduction remain unexplored or unreported. Historically, a gene knockout is the “gold standard” to determine whether a gene’s function is essential in vivo. Although knockout mice without apparent phenotypes are rarely published, these knockout mouse lines and their phenotypic information need to be shared to prevent redundant experiments. Herein, we used bioinformatic and experimental approaches to uncover mouse testis-enriched genes that are evolutionarilyconserved in humans. We then used gene-disruption approaches, including Knockout Mouse Project resources (targeting vectors and mice) and CRISPR/Cas9, to mutate and quickly analyze the fertility of these mutant mice. We discovered that 54 mutant mouse lines were fertile. Thus, despite evolutionary conservation of these genes in vertebrates and in some cases in all eukaryotes, our results indicate that these genes are not individually essential for male mouse fertility. Our phenotypic data are highly relevant in this fiscally tight funding period and postgenomic age when large numbers of genomes are being analyzed for disease association, and will prevent unnecessary expenditures and duplications of effort by others. PMID:27357688

Gene-expression analysis studies from Schultz et al. estimate that more than 2,300 genes in the mouse genome are expressed predominantly in the male germ line. As of their 2003 publication [Schultz N, Hamra FK, Garbers DL (2003) Proc Natl Acad Sci USA 100(21):12201-12206], the functions of the majority of these testis-enriched genes during spermatogenesis and fertilization were largely unknown. Since the study by Schultz et al., functional analysis of hundreds of reproductive-tract-enriched genes have been performed, but there remain many testis-enriched genes for which their relevance to reproduction remain unexplored or unreported. Historically, a gene knockout is the "gold standard" to determine whether a gene's function is essential in vivo. Although knockout mice without apparent phenotypes are rarely published, these knockout mouse lines and their phenotypic information need to be shared to prevent redundant experiments. Herein, we used bioinformatic and experimental approaches to uncover mouse testis-enriched genes that are evolutionarilyconserved in humans. We then used gene-disruption approaches, including Knockout Mouse Project resources (targeting vectors and mice) and CRISPR/Cas9, to mutate and quickly analyze the fertility of these mutant mice. We discovered that 54 mutant mouse lines were fertile. Thus, despite evolutionary conservation of these genes in vertebrates and in some cases in all eukaryotes, our results indicate that these genes are not individually essential for male mouse fertility. Our phenotypic data are highly relevant in this fiscally tight funding period and postgenomic age when large numbers of genomes are being analyzed for disease association, and will prevent unnecessary expenditures and duplications of effort by others.

Full Text Available Deacetylases of the Sir2 or sirtuin family are thought to regulate life cycle progression and life span in response to nutrient availability. This family has undergone successive rounds of duplication and diversification, enabling the enzymes to perform a wide variety of biological functions. Two evolutionarilyconserved functions of yeast Sir2 proteins are the generation of repressive chromatin in subtelomeric domains and the suppression of unbalanced recombination within the tandem rDNA array. Here, we describe the function of the Sir2 ortholog ClHst1 in the yeast Clavispora lusitaniae, an occasional opportunistic pathogen. ClHst1 was localized to the non-transcribed spacer regions of the rDNA repeats and deacetylated histones at these loci, indicating that, like other Sir2 proteins, ClHst1 modulates chromatin structure at the rDNA repeats. However, we found no evidence that ClHst1 associates with subtelomeric regions or impacts gene expression directly. This surprising observation highlights the plasticity of sirtuin function. Related yeast species, including Candida albicans, possess an additional Sir2 family member. Thus, it is likely that the ancestral Candida SIR2/HST1 gene was duplicated and subfunctionalized, such that HST1 retained the capacity to regulate rDNA whereas SIR2 had other functions, perhaps including the generation of subtelomeric chromatin. After subsequent species diversification, the SIR2 paralog was apparently lost in the C. lusitaniae lineage. Thus, C. lusitaniae presents an opportunity to discover how subtelomeric chromatin can be reconfigured.

Despite recent advances, it is yet not clear how intrinsically disordered regions in proteins recognize their targets without any defined structures. Short linear motifs had been proposed to mediate molecular recognition by disordered regions; however, the underlying structural prerequisite remains elusive. Moreover, the role of short linear motifs in DNA recognition has not been studied. We report a repertoire of short evolutionarilyConserved Recognition Elements (CoREs) in long intrinsically disordered regions, which have very distinct amino-acid propensities from those of known motifs, and exhibit a strong tendency to retain their three-dimensional conformations compared to adjacent regions. The majority of CoREs directly interact with the DNA in the available 3D structures, which is further supported by literature evidence, analyses of ΔΔG values of DNA-binding energies and threading-based prediction of DNA binding potential. CoREs were enriched in cancer-associated missense mutations, further strengthening their functional nature. Significant enrichment of glycines in CoREs and the preference of glycyl ϕ-Ψ values within the left-handed bridge range in the l-disallowed region of the Ramachandran plot suggest that Gly-to-nonGly mutations within CoREs might alter the backbone conformation and consequently the function, a hypothesis that we reconciled using available mutation data. We conclude that CoREs might serve as bait for DNA recognition by long disordered regions and that certain mutations in these peptides can disrupt their DNA binding potential and consequently the protein function. We further hypothesize that the preferred conformations of CoREs and of glycyl residues therein might play an important role in DNA binding. The highly ordered nature of CoREs hints at a therapeutic strategy to inhibit malicious molecular interactions using small molecules mimicking CoRE conformations.

SCN8A is a major neuronal sodium channel gene expressed throughout the central and peripheral nervous systems. Mutations of SCN8A result in movement disorders and impaired cognition. To investigate the basis for the tissue-specific expression of SCN8A, we located conserved, potentially regulatory sequences in the human, mouse, chicken, and fish genes by 5' RACE of brain RNA and genomic sequence comparison. A highly conserved 5' noncoding exon, exon 1c, is present in vertebrates from fish to mammals and appears to define the ancestral promoter region. The distance from exon 1c to the first coding exon increased tenfold during vertebrate evolution, largely by insertion of repetitive elements. The mammalian gene acquired three novel, mutually exclusive noncoding exons that are not represented in the lower vertebrates. Within the shared exon 1c, we identified four short sequence elements of 10-20 bp with an unusually high level of evolutionary conservation. The conserved elements are most similar to consensus sites for the transcription factors Pou6f1/Brn5, YY1, and REST/NRSF. Introduction of mutations into the predicted Pou6f1 and REST sites reduced promoter activity in transfected neuronal cells. A 470-bp promoter fragment containing all of the conserved elements directed brain-specific expression of the LacZ reporter in transgenic mice. Transgene expression was highest in hippocampal neurons and cerebellar Purkinje cells, consistent with the expression of the endogenous gene. The compact cluster of conserved regulatory elements in SCN8A provides a useful target for molecular analysis of neuronal gene expression.

Full Text Available The conserved DAF-16/FOXO transcription factors and SIR-2.1/SIRT1 deacetylases are critical for diverse biological processes, particularly longevity and stress response; and complex regulation of DAF-16/FOXO by SIR-2.1/SIRT1 is central to appropriate biological outcomes. Caenorhabditis elegans Host Cell Factor 1 (HCF-1 is a longevity determinant previously shown to act as a co-repressor of DAF-16. We report here that HCF-1 represents an integral player in the regulatory loop linking SIR-2.1/SIRT1 and DAF-16/FOXO in both worms and mammals. Genetic analyses showed that hcf-1 acts downstream of sir-2.1 to influence lifespan and oxidative stress response in C. elegans. Gene expression profiling revealed a striking 80% overlap between the DAF-16 target genes responsive to hcf-1 mutation and sir-2.1 overexpression. Subsequent GO-term analyses of HCF-1 and SIR-2.1-coregulated DAF-16 targets suggested that HCF-1 and SIR-2.1 together regulate specific aspects of DAF-16-mediated transcription particularly important for aging and stress responses. Analogous to its role in regulating DAF-16/SIR-2.1 target genes in C. elegans, the mammalian HCF-1 also repressed the expression of several FOXO/SIRT1 target genes. Protein-protein association studies demonstrated that SIR-2.1/SIRT1 and HCF-1 form protein complexes in worms and mammalian cells, highlighting the conservation of their regulatory relationship. Our findings uncover a conserved interaction between the key longevity determinants SIR-2.1/SIRT1 and HCF-1, and they provide new insights into the complex regulation of FOXO proteins.

The vitelline membrane (VM), the oocyte proximal layer of the Drosophila eggshell, contains four major proteins (VMPs) that possess a highly conserved “VM domain” which includes three precisely spaced, evolutionarilyconserved, cysteines (CX7CX8C). Focusing on sV23, this study showed that the three cysteines are not functionally equivalent. While substitution mutations at the first (C123S) or third (C140S) cysteines were tolerated, females with a substitution at the second position (C131S) we...

During evolution, enzyme-coding genes are acquired and/or replaced through lateral gene transfer and compiled into metabolic pathways. Gene regulatory networks evolve to fine tune biochemical fluxes through such metabolic pathways, enabling organisms to acclimate to nutrient fluctuations in a competitive environment. Here, we demonstrate that a single TrmB family transcription factor in Halobacterium salinarum NRC-1 globally coordinates functionally linked enzymes of diverse phylogeny in response to changes in carbon source availability. Specifically, during nutritional limitation, TrmB binds a cis-regulatory element to activate or repress 113 promoters of genes encoding enzymes in diverse metabolic pathways. By this mechanism, TrmB coordinates the expression of glycolysis, TCA cycle, and amino-acid biosynthesis pathways with the biosynthesis of their cognate cofactors (e.g. purine and thiamine). Notably, the TrmB-regulated metabolic network includes enzyme-coding genes that are uniquely archaeal as well as those that are conserved across all three domains of life. Simultaneous analysis of metabolic and gene regulatory network architectures suggests an ongoing process of co-evolution in which TrmB integrates the expression of metabolic enzyme-coding genes of diverse origins.

Mammals have co-evolved with retroviruses, including lentiviruses, over a long period. Evidence supporting this contention is that viral infectivity factor (Vif) encoded by lentiviruses antagonizes the anti-viral action of cellular apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3) of the host. To orchestrate E3 ubiquitin ligase complex for APOBEC3 degradation, Vifs utilize mammalian proteins such as core-binding factor beta (CBFB; for primate lentiviruses) or cyclophilin A (CYPA; for Maedi-Visna virus [MVV]). However, the co-evolutionary relationship between lentiviral Vif and the mammalian proteins associated with Vif-mediated APOBEC3 degradation is poorly understood. Moreover, it is unclear whether Vif proteins of small ruminant lentiviruses (SRLVs), including MVV and caprine arthritis encephalitis virus (CAEV), commonly utilize CYPA to degrade the APOBEC3 of their hosts. In this study, molecular phylogenetic and protein homology modeling revealed that Vif co-factors are evolutionarily and structurally conserved. It was also found that not only MVV but also CAEV Vifs degrade APOBEC3 of both sheep and goats and that CAEV Vifs interact with CYPA. These findings suggest that lentiviral Vifs chose evolutionarily and structurally stable proteins as their partners (e.g., CBFB or CYPA) for APOBEC3 degradation and, particularly, that SRLV Vifs evolved to utilize CYPA as their co-factor in degradation of ovine and caprine APOBEC3.

Full Text Available G-quadruplex DNA is a four-stranded DNA structure formed by non-Watson-Crick base pairing between stacked sets of four guanines. Many possible functions have been proposed for this structure, but its in vivo role in the cell is still largely unresolved. We carried out a genome-wide survey of the evolutionary conservation of regions with the potential to form G-quadruplex DNA structures (G4 DNA motifs across seven yeast species. We found that G4 DNA motifs were significantly more conserved than expected by chance, and the nucleotide-level conservation patterns suggested that the motif conservation was the result of the formation of G4 DNA structures. We characterized the association of conserved and non-conserved G4 DNA motifs in Saccharomyces cerevisiae with more than 40 known genome features and gene classes. Our comprehensive, integrated evolutionary and functional analysis confirmed the previously observed associations of G4 DNA motifs with promoter regions and the rDNA, and it identified several previously unrecognized associations of G4 DNA motifs with genomic features, such as mitotic and meiotic double-strand break sites (DSBs. Conserved G4 DNA motifs maintained strong associations with promoters and the rDNA, but not with DSBs. We also performed the first analysis of G4 DNA motifs in the mitochondria, and surprisingly found a tenfold higher concentration of the motifs in the AT-rich yeast mitochondrial DNA than in nuclear DNA. The evolutionary conservation of the G4 DNA motif and its association with specific genome features supports the hypothesis that G4 DNA has in vivo functions that are under evolutionary constraint.

While highly conserved through evolution, the cell cycle has been extensively modified to adapt to new developmental programs. Recently, analyses of mouse mutants revealed that several important cell cycle regulators are either dispensable for development or have a tissue- or cell-type-specific f...

The Miller-Dieker syndrome (MDS), composed of characteristic facial abnormalities and a severe neuronal migration disorder affecting the cerebral cortex, is caused by visible or submicroscopic deletions of chromosome band 17p13. Twelve anonymous DNA markers were tested against a panel of somatic cell hybrids containing 17p deletions from seven MDS patients. All patients, including three with normal karyotypes, are deleted for a variable set of 5-12 markers. Two highly polymorphic VNTR (variable number of tandem repeats) probes, YNZ22 and YNH37, are codeleted in all patients tested and make molecular diagnosis for this disorder feasible. By pulsed-field gel electrophoresis, YNZ22 and YNH37 were shown to be within 30 kilobases (kb) of each other. Cosmid clones containing both VNTR sequences were identified, and restriction mapping showed them to be <15 kb apart. Three overlapping cosmids spanning >100 kb were completely deleted in all patients, providing a minimum estimate of the size of the MDS critical region. A hypomethylated island and evolutionarilyconserved sequences were identified within this 100-kb region, indications of the presence of one or more expressed sequences potentially involved in the pathophysiology of this disorder. The conserved sequences were mapped to mouse chromosome 11 by using mouse-rat somatic cell hybrids, extending the remarkable homology between human chromosome 17 and mouse chromosome 11 by 30 centimorgans, into the 17p telomere region.

Because of N addition and variation in the site of VDJ joining, the third complementarity-determining region of the heavy chain (CDR-H3) is the most diverse component of the initial immunoglobulin antigen-binding site repertoire. A large component of the peritoneal cavity B-1 cell component is the product of fetal and perinatal B cell production. The CDR-H3 repertoire is thus depleted of N addition, which increases dependency on germ-line sequence. Cross-species comparisons have shown that DH gene sequence demonstrates conservation of amino acid preferences by reading frame. Preference for reading frame 1, which is enriched for tyrosine and glycine, is created both by rearrangement patterns and by pre-BCR and BCR selection. In previous studies, we have assessed the role of conserved DH sequence by examining peritoneal cavity B-1 cell numbers and antibody production in BALB/c mice with altered DH loci. Here, we review our finding that changes in the constraints normally imposed by germ-line-encoded amino acids within the CDR-H3 repertoire profoundly affect B-1 cell development, especially B-1a cells, and thus natural antibody immunity. Our studies suggest that both natural and somatic selection operate to create a restricted B-1 cell CDR-H3 repertoire.

Paired immunoglobulin-like receptor (PILR) α is an inhibitory receptor that recognizes several ligands, including mouse CD99, PILR-associating neural protein, and Herpes simplex virus-1 glycoprotein B. The physiological function(s) of interactions between PILRα and its cellular ligands are not well understood, as are the molecular determinants of PILRα/ligand interactions. To address these uncertainties, we sought to identify additional PILRα ligands and further define the molecular basis for PILRα/ligand interactions. Here, we identify two novel PILRα binding partners, neuronal differentiation and proliferation factor-1 (NPDC1), and collectin-12 (COLEC12). We find that sialylated O-glycans on these novel PILRα ligands, and on known PILRα ligands, are compulsory for PILRα binding. Sialylation-dependent ligand recognition is also a property of SIGLEC1, a member of the sialic acid-binding Ig-like lectins. SIGLEC1 Ig domain shares ∼22% sequence identity with PILRα, an identity that includes a conserved arginine localized to position 97 in mouse and human SIGLEC1, position 133 in mouse PILRα and position 126 in human PILRα. We observe that PILRα/ligand interactions require conserved PILRα Arg-133 (mouse) and Arg-126 (human), in correspondence with a previously reported requirement for SIGLEC1 Arg-197 in SIGLEC1/ligand interactions. Homology modeling identifies striking similarities between PILRα and SIGLEC1 ligand binding pockets as well as at least one set of distinctive interactions in the galactoxyl-binding site. Binding studies suggest that PILRα recognizes a complex ligand domain involving both sialic acid and protein motif(s). Thus, PILRα is evolved to engage multiple ligands with common molecular determinants to modulate myeloid cell functions in anatomical settings where PILRα ligands are expressed.

Full Text Available The novel protein Memo (Mediator of ErbB2 driven cell motility was identified in a screen for ErbB2 interacting proteins and found to have an essential function in cell motility. Memo is evolutionarilyconserved with homologs found in all branches of life; the human and yeast proteins have a similarity of >50%. In the present study we used the model organism S. cerevisiae to characterize the Memo-homologue Mho1 (Yjr008wp and to investigate its function in yeast. In a synthetic lethal screen we found MHO1 as a novel synthetic lethal partner of PLC1, which encodes the single phospholipase C in yeast. Double-deleted cells lacking MHO1 and PLC1, proliferate for up to ten generations. Introduction of human Memo into the memoΔplc1Δ strain rescued the synthetic lethal phenotype suggesting that yeast and human proteins have similar functions. Mho1 is present in the cytoplasm and the nucleus of yeast cells; the same distribution of Memo was found in mammalian cells. None of the Memo homologues have a characteristic nuclear localization sequence, however, a conserved nuclear export sequence is found in all. In mammalian cells, blocking nuclear export with Leptomycin B led to nuclear Memo accumulation, suggesting that it is actively exported from the nucleus. In yeast MHO1 expression is induced by stress conditions. Since invasive growth in S. cerevisiea is also stress-induced, we tested Mho1's role in this response. MHO1 deletion had no effect on invasion induced by nutrient deprivation, however, Mho1 overexpression blocked the invasive ability of yeast cells, suggesting that Mho1 might be acting in a dominant negative manner. Taken together, our results show that MHO1 is a novel synthetic lethal interactor with PLC1, and that both gene products are required for proliferation. Moreover, a role for Memo in cell motility/invasion appears to be conserved across species.

The temporal expression of ten clustered genes required for carotenoid (crt) and bacteriochlorophyll (bch) biosynthesis was examined during the transition from aerobic respiration to anaerobiosis requisite for the development of the photosynthetic membrane in the bacterium Rhodobacter capsulatus. Accumulation of crtA, crtC, crtD, crtE, crtF, crtK, bchC and bchD mRNAs increased transiently and coordinately, up to 12-fold following removal of oxygen from the growth medium, paralleling increases in mRNAs encoding pigment-binding polypeptides of the photosynthetic apparatus. The crtB and crtI genes, in contrast, were expressed similarly in the presence or absence of oxygen. The regulation patterns of promoters for the crtA and crtI genes and the bchCXYZ operon were characterized using lacZ transcriptional fusion and qualitatively reflected the corresponding mRNA accumulation patterns. We also report that the bchI gene product, encoded by a DNA sequence previously considered to be a portion of crtA, shares 49% sequence identity with the nuclear-encoded Arabidopsis thaliana Cs chloroplast protein required for normal pigmentation in plants.

Full Text Available Recent studies revealed new insights into the development of a unique caudal forebrain-signaling center: the zona limitans intrathalamica (zli. The zli is the last brain signaling center to form and the first forebrain compartment to be established. It is the only part of the dorsal neural tube expressing the morphogen Sonic Hedgehog (Shh whose activity participates in the survival, growth and patterning of neuronal progenitor subpopulations within the thalamic complex. Here, we review the gene regulatory network of transcription factors and cis-regulatory elements that underlies formation of a shh-expressing delimitated domain in the anterior brain. We discuss evidence that this network predates the origin of chordates. We highlight the contribution of Shh, Wnt and Notch signaling to zli development and discuss implications for the fact that the morphogen Shh relies on primary cilia for signal transduction. The network that underlies zli development also contributes to thalamus induction, and to its patterning once the zli has been set up. We present an overview of the brain malformations possibly associated with developmental defects in this gene regulatory network (GRN.

Full Text Available Abstract Background MicroRNAs (miRNAs play a fundamental role in the regulation of gene expression by translational repression or target mRNA degradation. Regulatory elements in miRNA promoters are less well studied, but may reveal a link between their expression and a specific cell type. Results To explore this link in myeloid cells, miRNA expression profiles were generated from monocytes and dendritic cells (DCs. Differences in miRNA expression among monocytes, DCs and their stimulated progeny were observed. Furthermore, putative promoter regions of miRNAs that are significantly up-regulated in DCs were screened for Transcription Factor Binding Sites (TFBSs based on TFBS motif matching score, the degree to which those TFBSs are over-represented in the promoters of the up-regulated miRNAs, and the extent of conservation of the TFBSs in mammals. Conclusions Analysis of evolutionarilyconserved TFBSs in DC promoters revealed preferential clustering of sites within 500 bp upstream of the precursor miRNAs and that many mRNAs of cognate TFs of the conserved TFBSs were indeed expressed in the DCs. Taken together, our data provide evidence that selected miRNAs expressed in DCs have evolutionarilyconserved TFBSs relevant to DC biology in their promoters.

The G protein-coupled receptor (GPCR) family is highly diversified and involved in many forms of information processing. SREB2 (GPR85) is the most conserved GPCR throughout vertebrate evolution and is expressed abundantly in brain structures exhibiting high levels of plasticity, e.g., the hippocampal dentate gyrus. Here, we show that SREB2 is involved in determining brain size, modulating diverse behaviors, and potentially in vulnerability to schizophrenia. Mild overexpression of SREB2 caused significant brain weight reduction and ventricular enlargement in transgenic (Tg) mice as well as behavioral abnormalities mirroring psychiatric disorders, e.g., decreased social interaction, abnormal sensorimotor gating, and impaired memory. SREB2 KO mice showed a reciprocal phenotype, a significant increase in brain weight accompanying a trend toward enhanced memory without apparent other behavioral abnormalities. In both Tg and KO mice, no gross malformation of brain structures was observed. Because of phenotypic overlap between SREB2 Tg mice and schizophrenia, we sought a possible link between the two. Minor alleles of two SREB2 SNPs, located in intron 2 and in the 3′ UTR, were overtransmitted to schizophrenia patients in a family-based sample and showed an allele load association with reduced hippocampal gray matter volume in patients. Our data implicate SREB2 as a potential risk factor for psychiatric disorders and its pathway as a target for psychiatric therapy. PMID:18413613

Full Text Available The Caenorhabditis elegans heterochronic gene pathway, which consists of a set of regulatory genes, plays an important regulatory role in the timing of stage-specific cell lineage development in nematodes. Research into the heterochronic gene pathway gave rise to landmark microRNA (miRNA studies and showed that these genes are important in stem cell and cancer biology; however, their functions in vertebrate development are largely unknown. To elucidate the function of the heterochronic gene pathway during vertebrate development, we cloned the zebrafish homologs of the C. elegans let-7 miRNA-binding protein, Lin-28, and analyzed their function in zebrafish development. The zebrafish genome contains two Lin28-related genes, lin-28a and lin-28b. Similar to mammalian Lin28 proteins, both zebrafish Lin-28a and Lin-28b have a conserved cold-shock domain and a pair of CCHC zinc finger domains, and are ubiquitously expressed during early embryonic development. In a reciprocal fashion, the expression of downstream heterochronic genes, let-7 and lin-4/miR-125 miRNA, occurred subsequent to lin-28 expression. The knockdown of Lin-28a or Lin-28b function by morpholino microinjection into embryos resulted in severe cell proliferation defects during early morphogenesis. We found that the expression of let-7 miRNA was upregulated and its downstream target gene, lin-41, was downregulated in these embryos. Interestingly, the expression of miR-430, a key regulator of maternal mRNA decay, was downregulated in lin-28a and lin-28b morphant embryos, suggesting a role for Lin-28 in the maternal-to-zygotic transition in zebrafish. Taken together, our results suggest an evolutionarilyconserved and pivotal role of the heterochronic gene pathway in early vertebrate embryogenesis.

The covalent attachment of ubiquitin to target proteins influences various cellular processes, including DNA repair, NF-kappaB signalling and cell survival. The most common mode of regulation by ubiquitin-conjugation involves specialized ubiquitin-binding proteins that bind to ubiquitylated proteins and link them to downstream biochemical processes. Unravelling how the ubiquitin-message is recognized is essential because aberrant ubiquitin-mediated signalling contributes to tumour formation. Recent evidence indicates that inhibitor of apoptosis (IAP) proteins are frequently overexpressed in cancer and their expression level is implicated in contributing to tumorigenesis, chemoresistance, disease progression and poor patient-survival. Here, we have identified an evolutionarilyconserved ubiquitin-associated (UBA) domain in IAPs, which enables them to bind to Lys 63-linked polyubiquitin. We found that the UBA domain is essential for the oncogenic potential of cIAP1, to maintain endothelial cell survival and to protect cells from TNF-alpha-induced apoptosis. Moreover, the UBA domain is required for XIAP and cIAP2-MALT1 to activate NF-kappaB. Our data suggest that the UBA domain of cIAP2-MALT1 stimulates NF-kappaB signalling by binding to polyubiquitylated NEMO. Significantly, 98% of all cIAP2-MALT1 fusion proteins retain the UBA domain, suggesting that ubiquitin-binding contributes to the oncogenic potential of cIAP2-MALT1 in MALT lymphoma. Our data identify IAPs as ubiquitin-binding proteins that contribute to ubiquitin-mediated cell survival, NF-kappaB signalling and oncogenesis.

The description of transcriptional regulatory networks has been pivotal in the understanding of operating principles under which organisms respond and adapt to varying conditions. While the study of the topology and dynamics of these networks has been the subject of considerable work, the investigation of the evolution of their topology, as a result of the adaptation of organisms to different environmental conditions, has received little attention. In this work, we study the evolution of transcriptional regulatory networks in bacteria from a genome reduction perspective, which manifests itself as the loss of genes at different degrees. We used the transcriptional regulatory network of Escherichia coli as a reference to compare 113 smaller, phylogenetically-related γ-proteobacteria, including 19 genomes of symbionts. We found that the type of regulatory action exerted by transcription factors, as genomes get progressively smaller, correlates well with their degree of conservation, with dual regulators being more conserved than repressors and activators in conditions of extreme reduction. In addition, we found that the preponderant conservation of dual regulators might be due to their role as both global regulators and nucleoid-associated proteins. We summarize our results in a conceptual model of how each TF type is gradually lost as genomes become smaller and give a rationale for the order in which this phenomenon occurs.

Full Text Available The description of transcriptional regulatory networks has been pivotal in the understanding of operating principles under which organisms respond and adapt to varying conditions. While the study of the topology and dynamics of these networks has been the subject of considerable work, the investigation of the evolution of their topology, as a result of the adaptation of organisms to different environmental conditions, has received little attention. In this work, we study the evolution of transcriptional regulatory networks in bacteria from a genome reduction perspective, which manifests itself as the loss of genes at different degrees. We used the transcriptional regulatory network of Escherichia coli as a reference to compare 113 smaller, phylogenetically-related γ-proteobacteria, including 19 genomes of symbionts. We found that the type of regulatory action exerted by transcription factors, as genomes get progressively smaller, correlates well with their degree of conservation, with dual regulators being more conserved than repressors and activators in conditions of extreme reduction. In addition, we found that the preponderant conservation of dual regulators might be due to their role as both global regulators and nucleoid-associated proteins. We summarize our results in a conceptual model of how each TF type is gradually lost as genomes become smaller and give a rationale for the order in which this phenomenon occurs.

Background As one of the most dominant bacterial groups on Earth, cyanobacteria play a pivotal role in the global carbon cycling and the Earth atmosphere composition. Understanding their molecular responses to environmental perturbations has important scientific and environmental values. Since important biological processes or networks are often evolutionarilyconserved, the cross-species transcriptional network analysis offers a useful strategy to decipher conserved and species-specific transcriptional mechanisms that cells utilize to deal with various biotic and abiotic disturbances, and it will eventually lead to a better understanding of associated adaptation and regulatory networks. Results In this study, the Weighted Gene Co-expression Network Analysis (WGCNA) approach was used to establish transcriptional networks for four important cyanobacteria species under metal stress, including iron depletion and high copper conditions. Cross-species network comparison led to discovery of several core response modules and genes possibly essential to metal stress, as well as species-specific hub genes for metal stresses in different cyanobacteria species, shedding light on survival strategies of cyanobacteria responding to different environmental perturbations. Conclusions The WGCNA analysis demonstrated that the application of cross-species transcriptional network analysis will lead to novel insights to molecular response to environmental changes which will otherwise not be achieved by analyzing data from a single species. PMID:23421563

Full Text Available Aging is the most significant risk factor for a range of diseases, including many cancers, neurodegeneration, cardiovascular disease, and diabetes. Caloric restriction (CR without malnutrition delays aging in diverse species, and therefore offers unique insights into age-related disease vulnerability. Previous studies suggest that there are shared mechanisms of disease resistance associated with delayed aging, however quantitative support is lacking. We therefore sought to identify a common response to CR in diverse tissues and species and determine whether this signature would reflect health status independent of aging. We analyzed gene expression datasets from eight tissues of mice subjected to CR and identified a common transcriptional signature that includes functional categories of mitochondrial energy metabolism, inflammation and ribosomal structure. This signature is detected in flies, rats, and rhesus monkeys on CR, indicating aspects of CR that are evolutionarilyconserved. Detection of the signature in mouse genetic models of slowed aging indicates that it is not unique to CR but rather a common aspect of extended longevity. Mice lacking the NAD-dependent deacetylase SIRT3 fail to induce mitochondrial and anti-inflammatory elements of the signature in response to CR, suggesting a potential mechanism involving SIRT3. The inverse of this transcriptional signature is detected with consumption of a high fat diet, obesity and metabolic disease, and is reversed in response to interventions that decrease disease risk. We propose that this evolutionarilyconserved, tissue-independent, transcriptional signature of delayed aging and reduced disease vulnerability is a promising target for developing therapies for age-related diseases.

Nuclear-mitochondrial gene interactions governing cytoplasmic male sterility (CMS) in angiosperms have been found to be unique to each system. Fertility restoration of three diverse alloplasmic CMS lines of Brassica juncea by a line carrying the fertility-restorer gene introgressed from Moricandia arvensis prompted this investigation to examine the molecular basis of CMS in these lines. Since previous studies had found altered atpA transcription associated with CMS in these lines, the atpA genes and transcripts of CMS, fertility-restored, and euplasmic lines were cloned and compared. atpA coding and downstream sequences were conserved among CMS and euplasmic lines but major differences were found in the 5' flanking sequences of atpA. A unique open reading frame (ORF), orf108, co-transcribed with atpA, was found in male sterile flowers of CMS lines carrying mitochondrial genomes of Diplotaxis berthautii, D. catholica, or D. erucoides. In presence of the restorer gene, the bicistronic orf108-atpA transcript was cleaved within orf108 to yield a monocistronic atpA transcript. Transgenic expression of orf108 with anther-specific Atprx18 promoter in Arabidopsis thaliana gave 50% pollen sterility, indicating that Orf108 is lethal at the gametophytic stage. Further, lack of transmission of orf108 to the progeny showed for the first time that mitochondrial ORFs could also cause female sterility. orf108 was found to be widely distributed among wild relatives of Brassica, indicating its ancient origin. This is the first report that shows that CMS lines of different origin and morphology could share common molecular basis. The gametic lethality of Orf108 offers a novel opportunity for transgene containment.

Hemocytes are the effective immunocytes in bivalves, which have been reported to be derived from stem-like cells in gill epithelium of oyster. In the present work, a conserved haematopoietic transcription factor Tal-1/Scl (Stem Cell Leukemia) was identified in Pacific oyster (Cg-SCL), and it was evolutionarily close to the orthologs in deuterostomes. Cg-SCL was highly distributed in the hemocytes as well as gill and mantle. The hemocyte specific genes Integrin, EcSOD and haematopoietic transcription factors GATA3, C-Myb, c-kit, were down-regulated when Cg-SCL was interfered by dsRNA. During the larval developmental stages, the mRNA transcripts of Cg-SCL gradually increased after fertilization and peaked at early trochophore larvae stage (10 hpf, hours post fertilization), then sharply decreased in late trochophore larvae stage (15 hpf) before resuming in umbo larvae (120 hpf). Whole-mount immunofluorescence assay further revealed that the immunoreactivity of Cg-SCL appeared in blastula larvae with two approximate symmetric spots, and this expression pattern lasted in gastrula larvae. By trochophore, the immunoreactivity formed a ring around the dorsal region and then separated into two remarkable spots at the dorsal side in D-veliger larvae. After bacterial challenge, the mRNA expression levels of Cg-SCL were significantly up-regulated in the D-veliger and umbo larvae, indicating the available hematopoietic regulation in oyster larvae. These results demonstrated that Cg-SCL could be used as haematopoietic specific marker to trace potential developmental events of hematopoiesis during ontogenesis of oyster, which occurred early in blastula stage and maintained until D-veliger larvae.

Mapping the DNA-binding preferences of transcription factor (TF) complexes is critical for deciphering the functions of cis-regulatory elements. Here, we developed a computational method that compares co-occurring motif spacings in conserved versus unconserved regions of the human genome to detect evolutionarily constrained binding sites of rigid TF complexes. Structural data were used to estimate TF complex physical plausibility, explore overlapping motif arrangements seldom tackled by non-structure-aware methods, and generate and analyse three-dimensional models of the predicted complexes bound to DNA. Using this approach, we predicted 422 physically realistic TF complex motifs at 18% false discovery rate, the majority of which (326, 77%) contain some sequence overlap between binding sites. The set of mostly novel complexes is enriched in known composite motifs, predictive of binding site configurations in TF-TF-DNA crystal structures, and supported by ChIP-seq datasets. Structural modelling revealed three cooperativity mechanisms: direct protein-protein interactions, potentially indirect interactions and \\'through-DNA\\' interactions. Indeed, 38% of the predicted complexes were found to contain four or more bases in which TF pairs appear to synergize through overlapping binding to the same DNA base pairs in opposite grooves or strands. Our TF complex and associated binding site predictions are available as a web resource at http://bejerano.stanford.edu/complex.

Mouse models have been used extensively for decades and have been instrumental in improving our understanding of mammalian erythropoiesis. Nonetheless, there are several examples of variation between human and mouse erythropoiesis. We performed a comparative global gene expression study using data from morphologically identical stage-matched sorted populations of human and mouse erythroid precursors from early to late erythroblasts. Induction and repression of major transcriptional regulators of erythropoiesis, as well as major erythroid-important proteins, are largely conserved between the species. In contrast, at a global level we identified a significant extent of divergence between the species, both at comparable stages and in the transitions between stages, especially for the 500 most highly expressed genes during development. This suggests that the response of multiple developmentally regulated genes to key erythroid transcriptional regulators represents an important modification that has occurred in the course of erythroid evolution. In developing a systematic framework to understand and study conservation and divergence between human and mouse erythropoiesis, we show how mouse models can fail to mimic specific human diseases and provide predictions for translating findings from mouse models to potential therapies for human disease.

To investigate the role of Hippo pathway signaling during vertebrate development transgenic zebrafish lines were generated and validated to dynamically monitor and manipulate Yap/Taz-Tead activity. Spatial and temporal analysis of Yap/Taz-Tead activity suggested the importance of Hippo signaling during cardiac precursor migration and other developmental processes. When the transcriptional co-activators, Yap and Taz were restricted from interacting with DNA-binding Tead transcription factors through expression of a dominant negative transgene, cardiac precursors failed to migrate completely to the midline resulting in strong cardia bifida. Yap/Taz-Tead activity reporters also allowed us to investigate upstream and downstream factors known to regulate Hippo signaling output in Drosophila. While Crumbs mutations in Drosophila eye disc epithelia increase nuclear translocation and activity of Yorkie (the fly homolog of Yap/Taz), zebrafish crb2a mutants lacked nuclear Yap positive cells and down-regulated Yap/Taz-Tead activity reporters in the eye epithelia, despite the loss of apical-basal cell polarity in those cells. However, as an example of evolutionary conservation, the Tondu-domain containing protein Vestigial-like 4b (Vgll4b) was found to down-regulate endogenous Yap/Taz-Tead activity in the retinal pigment epithelium, similar to Drosophila Tgi in imaginal discs. In conclusion, the Yap/Taz-Tead activity reporters revealed the dynamics of Yap/Taz-Tead signaling and novel insights into Hippo pathway regulation for vertebrates. These studies highlight the utility of this transgenic tool-suite for ongoing analysis into the mechanisms of Hippo pathway regulation and the consequences of signaling output.

Full Text Available The zinc finger transcription factor CONSTANS has a well-established central role in the mechanism for photoperiod sensing in Arabidopsis, integrating light and circadian clock signals to upregulate the florigen gene FT under long-day but not short-day conditions. Although CONSTANS-like (COL genes in other species have also been shown to regulate flowering time, it is not clear how widely this central role in photoperiod sensing is conserved.Legumes are a major plant group and various legume species show significant natural variation for photoperiod responsive flowering. Orthologs of several Arabidopsis genes have been shown to participate in photoperiodic flowering in legumes, but the possible function of COL genes as integrators of the photoperiod response has not yet been examined in detail. Here we characterize the COL family in the temperate long-day legume Medicago truncatula, using expression analyses, reverse genetics, transient activation assays and Arabidopsis transformation. Our results provide several lines of evidence suggesting that COL genes are unlikely to have a central role in the photoperiod response mechanism in this species.

Gene expression variation between species is a major contributor to phenotypic diversity, yet the underlying flexibility of transcriptional regulatory networks remains largely unexplored. Transcription of the ribosomal regulon is a critical task for all cells; in S. cerevisiae the transcription factors Rap1, Fhl1, Ifh1, and Hmo1 form a multi-subunit complex that controls ribosomal gene expression, while in C. albicans this regulation is under the control of Tbf1 and Cbf1. Here, we analyzed, u...

The oil palm (Elaeis guineensis), a monocotyledonous species in the family Arecaceae, has an extraordinarily oil rich fleshy mesocarp, and presents an original model to examine the ripening processes and regulation in this particular monocot fruit. Histochemical analysis and cell parameter measurements revealed cell wall and middle lamella expansion and degradation during ripening and in response to ethylene. Cell wall related transcript profiles suggest a transition from synthesis to degradation is under transcriptional control during ripening, in particular a switch from cellulose, hemicellulose, and pectin synthesis to hydrolysis and degradation. The data provide evidence for the transcriptional activation of expansin, polygalacturonase, mannosidase, beta-galactosidase, and xyloglucan endotransglucosylase/hydrolase proteins in the ripening oil palm mesocarp, suggesting widespread conservation of these activities during ripening for monocotyledonous and eudicotyledonous fruit types. Profiling of the most abundant oil palm polygalacturonase (EgPG4) and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) transcripts during development and in response to ethylene demonstrated both are sensitive markers of ethylene production and inducible gene expression during mesocarp ripening, and provide evidence for a conserved regulatory module between ethylene and cell wall pectin degradation. A comprehensive analysis of NAC transcription factors confirmed at least 10 transcripts from diverse NAC domain clades are expressed in the mesocarp during ripening, four of which are induced by ethylene treatment, with the two most inducible (EgNAC6 and EgNAC7) phylogenetically similar to the tomato NAC-NOR master-ripening regulator. Overall, the results provide evidence that despite the phylogenetic distance of the oil palm within the family Arecaceae from the most extensively studied monocot banana fruit, it appears ripening of divergent monocot and eudicot fruit lineages are

The Arp2/3 protein complex has been implicated in the control of actin polymerization in cells. The human complex consists of seven subunits which include the actin related proteins Arp2 and Arp3, and five others referred to as p41-Arc, p34-Arc, p21-Arc, p20-Arc, and p16-Arc (Arp complex). We have determined the predicted amino acid sequence of all seven subunits. Each has homologues in diverse eukaryotes, implying that the structure and function of the complex has been conserved through evol...

Full Text Available Arginine-rich tandem zinc-finger proteins (RR-TZF participate in a wide range of plant developmental processes and adaptive responses to abiotic stress, such as cold, salt and drought. This study investigates the conservation of the genes AtTZF1-5 at the level of their sequences and expression across plant species. The genomic sequences of the two RR-TZF genes TdTZF1-A and TdTZF1-B were isolated in durum wheat and assigned to chromosomes 3A and 3B, respectively. Sequence comparisons revealed that they encode proteins that are highly homologous to AtTZF1, AtTZF2 and AtTZF3. The expression profiles of these RR-TZF durum wheat and Arabidopsis proteins support a common function in the regulation of seed germination and responses to abiotic stress. In particular, analysis of plants with attenuated and overexpressed AtTZF3 indicate that AtTZF3 is a negative regulator of seed germination under conditions of salt stress. Finally, comparative sequence analyses establish that the RR-TZF genes are encoded by lower plants, including the bryophyte Physcomitrella patens and the alga Chlamydomonas reinhardtii. The regulation of the Physcomitrella AtTZF1-2-3-like genes by salt stress strongly suggests that a subgroup of the RR-TZF proteins has a function that has been conserved throughout evolution.

The dopaminergic system influences motor behavior, signals reward and novelty, and is an essential component of the basal ganglia in all vertebrates including the lamprey, one of the phylogenetically oldest vertebrates. The intrinsic organization and function of the lamprey basal ganglia is highly conserved. For instance, the direct and indirect pathways are modulated through dopamine D1 and D2 receptors in lamprey and in mammals. The nucleus of the tuberculum posterior, a homologue of the substantia nigra pars compacta (SNc)/ventral tegmental area (VTA) is present in lamprey, but only scarce data exist about its connectivity. Likewise, the D2 receptor is expressed in the striatum, but little is known about its localization in other brain areas. We used in situ hybridization and tracer injections, both in combination with tyrosine hydroxylase immunohistochemistry, to characterize the SNc/VTA efferent and afferent connectivity, and to relate its projection pattern with D2 receptor expression in particular. We show that most features of the dopaminergic system are highly conserved. As in mammals, the direct pallial (cortex in mammals) input and the basal ganglia connectivity with the SNc/VTA are present as part of the evaluation system, as well as input from the tectum as the evolutionary basis for salience/novelty detection. Moreover, the SNc/VTA receives sensory information from the olfactory bulbs, optic tectum, octavolateral area, and dorsal column nucleus, and it innervates, apart from the nigrostriatal pathway, several motor-related areas. This suggests that the dopaminergic system also contributes to the control of different motor centers at the brainstem level.

The early evolution of plants required the acquisition of a number of key adaptations to overcome physiological difficulties associated with survival on land. One of these was a tough sporopollenin wall that enclosed reproductive propagules and provided protection from desiccation and UV-B radiation. All land plants possess such walled spores (or their derived homologue, pollen). We took a reverse genetics approach, consisting of knock-out and complementation experiments to test the functional conservation of the sporopollenin-associated gene MALE STERILTY 2 (which is essential for pollen wall development in Arabidopsis thaliana) in the bryophyte Physcomitrella patens. Knock-outs of a putative moss homologue of the A. thaliana MS2 gene, which is highly expressed in the moss sporophyte, led to spores with highly defective walls comparable to that observed in the A. thaliana ms2 mutant, and extremely compromised germination. Conversely, the moss MS2 gene could not rescue the A. thaliana ms2 phenotype. The results presented here suggest that a core component of the biochemical and developmental pathway required for angiosperm pollen wall development was recruited early in land plant evolution but the continued increase in pollen wall complexity observed in angiosperms has been accompanied by divergence in MS2 gene function.

The tumor suppressor protein programmed cell death 4 (Pdcd4) has been implicated in the translational regulation of specific mRNAs, however, the identities of the natural Pdcd4 target mRNAs and the mechanisms by which Pdcd4 affects their translation are not well understood. Pdcd4 binds to the eukaryotic translation initiation factor eIF4A and inhibits its helicase activity, which has suggested that Pdcd4 suppresses translation initiation of mRNAs containing structured 5'-untranslated regions. Recent work has revealed a second inhibitory mechanism, which is eIF4A-independent and involves direct RNA-binding of Pdcd4 to the target mRNAs. We have now identified the poly(A)-binding protein (PABP) as a novel direct interaction partner of Pdcd4. The ability to interact with PABP is shared between human and Drosophila Pdcd4, indicating that it has been highly conserved during evolution. Mutants of Pdcd4 that have lost the ability to interact with PABP fail to stably associate with ribosomal complexes in sucrose density gradients and to suppress translation, as exemplified by c-myb mRNA. Overall, our work identifies PABP as a novel functionally relevant Pdcd4 interaction partner that contributes to the regulation of translation by Pdcd4.

We predicted in human peroxisomal membrane proteins (PMPs) the binding sites for PEX19, a key player in the topogenesis of PMPs, by virtue of an algorithm developed for yeast PMPs. The best scoring PEX19-binding site was found in the adrenoleukodystrophy protein (ALDP). The identified site was indeed bound by human PEX19 and was also recognized by the orthologous yeast PEX19 protein. Likewise, both human and yeast PEX19 bound with comparable affinities to the PEX19-binding site of the yeast PMP Pex13p. Interestingly, the identified PEX19-binding site of ALDP coincided with its previously determined targeting motif. We corroborated the requirement of the ALDP PEX19-binding site for peroxisomal targeting in human fibroblasts and showed that the minimal ALDP fragment targets correctly also in yeast, again in a PEX19-binding site-dependent manner. Furthermore, the human PEX19-binding site of ALDP proved interchangeable with that of yeast Pex13p in an in vivo targeting assay. Finally, we showed in vitro that most of the predicted binding sequences of human PMPs represent true binding sites for human PEX19, indicating that human PMPs harbor common PEX19-binding sites that do resemble those of yeast. Our data clearly revealed a role for PEX19-binding sites as PMP-targeting motifs across species, thereby demonstrating the evolutionary conservation of PMP signal sequences from yeast to man.

In seed plants, strigolactones (SLs) regulate architecture and induce mycorrhizal symbiosis in response to environmental cues. SLs are formed by combined activity of the carotenoid cleavage dioxygenases (CCDs) 7 and 8 from 9-cis-β-carotene, leading to carlactone that is converted by cytochromes P450 (clade 711; MAX1 in Arabidopsis) into various SLs. As Physcomitrella patens possesses CCD7 and CCD8 homologs but lacks MAX1, we investigated if PpCCD7 together with PpCCD8 form carlactone and how deletion of these enzymes influences growth and interactions with the environment. We investigated the enzymatic activity of PpCCD7 and PpCCD8 in vitro, identified the formed products by high performance liquid chromatography (HPLC) and LC-MS, and generated and analysed ΔCCD7 and ΔCCD8 mutants. We defined enzymatic activity of PpCCD7 as a stereospecific 9-cis-CCD and PpCCD8 as a carlactone synthase. ΔCCD7 and ΔCCD8 lines showed enhanced caulonema growth, which was revertible by adding the SL analogue GR24 or carlactone. Wild-type (WT) exudates induced seed germination in Orobanche ramosa. This activity was increased upon phosphate starvation and abolished in exudates of both mutants. Furthermore, both mutants showed increased susceptibility to phytopathogenic fungi. Our study reveals the deep evolutionary conservation of SL biosynthesis, SL function, and its regulation by biotic and abiotic cues.

Full Text Available Abstract Background Ticks are vectors for a variety of viral, bacterial and parasitic diseases in human and domestic animals. To survive and reproduce ticks feed on host blood, yet our understanding of the intestinal proteolytic machinery used to derive absorbable nutrients from the blood meal is poor. Intestinal digestive processes are limiting factors for pathogen transmission since the tick gut presents the primary site of infection. Moreover, digestive enzymes may find practical application as anti-tick vaccine targets. Results Using the hard tick, Ixodes ricinus, we performed a functional activity scan of the peptidase complement in gut tissue extracts that demonstrated the presence of five types of peptidases of the cysteine and aspartic classes. We followed up with genetic screens of gut-derived cDNA to identify and clone genes encoding the cysteine peptidases cathepsins B, L and C, an asparaginyl endopeptidase (legumain, and the aspartic peptidase, cathepsin D. By RT-PCR, expression of asparaginyl endopeptidase and cathepsins B and D was restricted to gut tissue and to those developmental stages feeding on blood. Conclusion Overall, our results demonstrate the presence of a network of cysteine and aspartic peptidases that conceivably operates to digest host blood proteins in a concerted manner. Significantly, the peptidase components of this digestive network are orthologous to those described in other parasites, including nematodes and flatworms. Accordingly, the present data and those available for other tick species support the notion of an evolutionary conservation of a cysteine/aspartic peptidase system for digestion that includes ticks, but differs from that of insects relying on serine peptidases.

Lipid droplets, which are conserved across almost all species, are cytoplasmic organelles used to store neutral lipids. Identification of lipid droplet regulators will be conducive to resolving obesity and other fat-associated diseases. In this paper, we selected 11 candidates that might be associated with lipid metabolism in Caenorhabditis elegans. Using a BODIPY 493/503-based flow cytometry screen, 6 negative and 3 positive regulators of fat content were identified. We selected one negative regulator of lipid content, C13C4.5, for future study. C13C4.5 was mainly expressed in the worm intestine. We found that this gene was important for maintaining the metabolism of lipid droplets. Biochemical results revealed that 50% of triacylglycerol (TAG) was lost in C13C4.5 knockout worms. Stimulated Raman scattering (SRS) signals in C13C4.5 mutants showed only 49.6% of the fat content in the proximal intestinal region and 86.3% in the distal intestinal region compared with wild type animals. The mean values of lipid droplet size and intensity in C13C4.5 knockout animals were found to be significantly decreased compared with those in wild type worms. The LMP-1-labeled membrane structures in worm intestines were also enlarged in C13C4.5 mutant animals. Finally, fertility defects were found in C13C4.5(ok2087) mutants. Taken together, these results indicate that C13C4.5 may regulate the fertility of C. elegans by changing the size and fat content of lipid droplets by interfering with lysosomal morphology and function.

The Arp2/3 protein complex has been implicated in the control of actin polymerization in cells. The human complex consists of seven subunits which include the actin related proteins Arp2 and Arp3, and five others referred to as p41-Arc, p34-Arc, p21-Arc, p20-Arc, and p16-Arc (p omplex). We have determined the predicted amino acid sequence of all seven subunits. Each has homologues in diverse eukaryotes, implying that the structure and function of the complex has been conserved through evolution. Human Arp2 and Arp3 are very similar to family members from other species. p41-Arc is a new member of the Sop2 family of WD (tryptophan and aspartate) repeat-containing proteins and may be posttranslationally modified, suggesting that it may be involved in regulating the activity and/or localization of the complex. p34-Arc, p21-Arc, p20-Arc, and p16-Arc define novel protein families. We sought to evaluate the function of the Arp2/3 complex in cells by determining its intracellular distribution. Arp3, p34-Arc, and p21-Arc were localized to the lamellipodia of stationary and locomoting fibroblasts, as well to Listeria monocytogenes assembled actin tails. They were not detected in cellular bundles of actin filaments. Taken together with the ability of the Arp2/3 complex to induce actin polymerization, these observations suggest that the complex promotes actin assembly in lamellipodia and may participate in lamellipodial protrusion.

Highlights: •GCN1 is required for mammalian and yeast GCN2 function in a variety of conditions. •Mammalian IMPACT competes with GCN2 for GCN1 binding. •IMPACT and its yeast counterpart YIH1 downregulate GCN1-dependent GCN2 activation. -- Abstract: In response to a range of environmental stresses, phosphorylation of the alpha subunit of the translation initiation factor 2 (eIF2α) represses general protein synthesis coincident with increased translation of specific mRNAs, such as those encoding the transcription activators GCN4 and ATF4. The eIF2α kinase GCN2 is activated by amino acid starvation by a mechanism involving GCN2 binding to an activator protein GCN1, along with association with uncharged tRNA that accumulates during nutrient deprivation. We previously showed that mammalian IMPACT and its yeast ortholog YIH1 bind to GCN1, thereby preventing GCN1 association with GCN2 and stimulation of this eIF2α kinase during amino acid depletion. GCN2 activity is also enhanced by other stresses, including proteasome inhibition, UV irradiation and lack of glucose. Here, we provide evidence that IMPACT affects directly and specifically the activation of GCN2 under these stress conditions in mammalian cells. We show that activation of mammalian GCN2 requires its interaction with GCN1 and that IMPACT promotes the dissolution of the GCN2–GCN1 complex. To a similar extent as the overexpression of YIH1, overexpression of IMPACT in yeast cells inhibited growth under all stress conditions that require GCN2 and GCN1 for cell survival, including exposure to acetic acid, high levels of NaCl, H{sub 2}O{sub 2} or benomyl. This study extends our understanding of the roles played by GCN1 in GCN2 activation induced by a variety of stress arrangements and suggests that IMPACT and YIH1 use similar mechanisms for regulating this eIF2α kinase.

Box C/D ribonucleoprotein particles guide the 2'-O-ribose methylation of target nucleotides in both archaeal and eukaryotic RNAs. These complexes contain two functional centers, assembled around the C/D and C'/D' motifs in the box C/D RNA. The C/D and C'/D' RNPs of the archaeal snoRNA-like RNP (sRNP) are spatially and functionally coupled. Here, we show that similar coupling also occurs in eukaryotic box C/D snoRNPs. The C/D RNP guided 2'-O-methylation when the C'/D' motif was either mutated or ablated. In contrast, the C'/D' RNP was inactive as an independent complex. Additional experiments demonstrated that the internal C'/D' RNP is spatially coupled to the terminal box C/D complex. Pulldown experiments also indicated that all four core proteins are independently recruited to the box C/D and C'/D' motifs. Therefore, the spatial-functional coupling of box C/D and C'/D' RNPs is an evolutionarilyconserved feature of both archaeal and eukaryotic box C/D RNP complexes.

The activity of transcription factors is usually governed by allosteric physicochemical signals or metabolites, which are in turn produced in the cell or obtained from the environment by the activity of the products of effector genes. Previously, we identified a collection of more than 110 transcription factors and their corresponding effector genes in Escherichia coli K-12. Here, we introduce the notion of "triferog", which relates to the identification of orthologous transcription factors and effector genes across genomes and show that transcriptional sensing systems known in E. coli are poorly conserved beyond Salmonella. We also find that enzymes that act as effector genes for the production of endogenous effector metabolites are more conserved than their corresponding effector genes encoding for transport and two-component systems for sensing exogenous signals. Finally, we observe that on an evolutionary scale enzymes are more conserved than their respective TFs, suggesting a homogenous cellular metabolism across genomes and the conservation of transcriptional control of critical cellular processes like DNA replication by a common endogenous signal. We hypothesize that extensive variation in the domain architecture of TFs and changes in endogenous conditions at large phylogenetic distances could be the major contributing factors for the observed differential conservation of TFs and their corresponding effector genes encoding for enzymes, causing variations in transcriptional responses across organisms.

Full Text Available Abstract Background DNA methylation can regulate gene expression by modulating the interaction between DNA and proteins or protein complexes. Conserved consensus motifs exist across the human genome ("predicted transcription factor binding sites": "predicted TFBS" but the large majority of these are proven by chromatin immunoprecipitation and high throughput sequencing (ChIP-seq not to be biological transcription factor binding sites ("empirical TFBS". We hypothesize that DNA methylation at conserved consensus motifs prevents promiscuous or disorderly transcription factor binding. Results Using genome-wide methylation maps of the human heart and sperm, we found that all conserved consensus motifs as well as the subset of those that reside outside CpG islands have an aggregate profile of hyper-methylation. In contrast, empirical TFBS with conserved consensus motifs have a profile of hypo-methylation. 40% of empirical TFBS with conserved consensus motifs resided in CpG islands whereas only 7% of all conserved consensus motifs were in CpG islands. Finally we further identified a minority subset of TF whose profiles are either hypo-methylated or neutral at their respective conserved consensus motifs implicating that these TF may be responsible for establishing or maintaining an un-methylated DNA state, or whose binding is not regulated by DNA methylation. Conclusions Our analysis supports the hypothesis that at least for a subset of TF, empirical binding to conserved consensus motifs genome-wide may be controlled by DNA methylation.

Quiescence is a conserved cell-cycle state characterized by cell-cycle arrest, increased stress resistance, enhanced longevity, and decreased transcriptional, translational, and metabolic output. Although quiescence plays essential roles in cell survival and normal differentiation, the molecular mechanisms leading to this state are not well understood. Here, we determined changes in the transcriptome and chromatin structure of S. cerevisiae upon quiescence entry. Our analyses revealed transcriptional shutoff that is far more robust than previously believed and an unprecedented global chromatin transition, which are tightly correlated. These changes require Rpd3 lysine deacetylase targeting to at least half of gene promoters via quiescence-specific transcription factors including Xbp1 and Stb3. Deletion of RPD3 prevents cells from establishing transcriptional quiescence, leading to defects in quiescence entry and shortening of chronological lifespan. Our results define a molecular mechanism for global reprogramming of transcriptome and chromatin structure for quiescence driven by a highly conserved chromatin regulator.

The DNA sequence motif ATTTGCAT (octamer) or its inverse complement has been identified as an evolutionarilyconserved element in the promoter region of immunoglobulin genes. Two major DNA-binding proteins that bind in a sequence-specific manner to the octamer DNA sequence have been identified in mammalian species--a ubiquitously expressed protein (Oct-1) and a lymphoid-specific protein (Oct-2). During characterization of the promoter region of the chicken immunoglobulin light chain gene, the authors identified two homologous octamer-binding proteins in chicken B cells. when the cloning of the human gene for Oct-2 revealed it to be a member of a distinct family of homeobox genes, they sought to determine if the human Oct-2 cDNA could be used to identify homologous chicken homeobox genes. Using a human Oct-2 homeobox-specific DNA probe, they were able to identify 6-10 homeobox-containing genes in the chicken genome, demonstrating that the Oct-2-related subfamily of homeobox genes exists in avian species. DNA sequence analysis revealed it to be the chicken homologue of the human Oct-1 gene. Together, the data show that the POU-containing subfamily of homeobox genes have been highly conserved during vertebrate evolution, apparently as a result of selection for their DNA-binding and transcriptional regulatory properties.

Full Text Available Ultra-conserved genes or elements (UCGs/UCEs in the human genome are extreme examples of conservation. We characterized natural variations in 2884 UCEs and UCGs in two distinct populations; Singaporean Chinese (n = 280 and Italian (n = 501 by using a pooled sample, targeted capture, sequencing approach. We identify, with high confidence, in these regions the abundance of rare SNVs (MAF5% are more often found in relatively less-conserved nucleotides within UCEs, compared to rare variants. Moreover, prevalent variants are less likely to overlap transcription factor binding site. Using SNPfold we found no significant influence of RNA secondary structure on UCE conservation. All together, these results suggest UCEs are not under selective pressure as a stretch of DNA but are under differential evolutionary pressure on the single nucleotide level.

HNULP1,a new member of the basic helixloop-helix transcription factors,contains a DUF654 domain in its C-terminus and is highly conserved from Drosophilae,yeast,zebrafish to mouse.The function of this motif,however,is currently unknown.In this research,we fused five deletion fragments of the DUF654 domain to the GAL4 DNA-binding domain and then co-transfected with plasmids L8G5-Luc and VP-16.The analysis of the GAL4 luciferase reporter gene indicated that fragments from 228 to 407 amino acids in the DUF654 domain had a strong transcription repression activity.Therefore,this study lays a solid foundation for research on the mechanism of hnulp1 transcriptional regulation and the function of the DUF654 domain.

Full Text Available Abstract Background Increasing evidence shows that whole genomes of eukaryotes are almost entirely transcribed into both protein coding genes and an enormous number of non-protein-coding RNAs (ncRNAs. Therefore, revealing the underlying regulatory mechanisms of transcripts becomes imperative. However, for a complete understanding of transcriptional regulatory mechanisms, we need to identify the regions in which they are found. We will call these transcriptional regulation regions, or TRRs, which can be considered functional regions containing a cluster of regulatory elements that cooperatively recruit transcriptional factors for binding and then regulating the expression of transcripts. Results We constructed a hierarchical stochastic language (HSL model for the identification of core TRRs in yeast based on regulatory cooperation among TRR elements. The HSL model trained based on yeast achieved comparable accuracy in predicting TRRs in other species, e.g., fruit fly, human, and rice, thus demonstrating the conservation of TRRs across species. The HSL model was also used to identify the TRRs of genes, such as p53 or OsALYL1, as well as microRNAs. In addition, the ENCODE regions were examined by HSL, and TRRs were found to pervasively locate in the genomes. Conclusion Our findings indicate that 1 the HSL model can be used to accurately predict core TRRs of transcripts across species and 2 identified core TRRs by HSL are proper candidates for the further scrutiny of specific regulatory elements and mechanisms. Meanwhile, the regulatory activity taking place in the abundant numbers of ncRNAs might account for the ubiquitous presence of TRRs across the genome. In addition, we also found that the TRRs of protein coding genes and ncRNAs are similar in structure, with the latter being more conserved than the former.

Results from a previous rice transcription mapping and the GeSTer algorithm analysis used in this investigation for rice plastid genome suggest that an inverted repeat, IRs18, in 3' region of the plastid rps18 gene may serve as a transcription terminator. The in vitro transcription assay showed that the transcript ending at the IRs18 was not processed by ribonucleases but terminated intrinsically in an rNTP substrate-dependent manner as demonstrated for the first time in plant gene regulation. For the poly-T tract (TTCTTTTTT) 3'-proximal to the IRs18, the C base conversion to T resulting in a perfect 9 Ts can dramatically increase termination efficiency, which is a common feature of bacterial intrinsic termination. This study is the first case to indicate that a conserved inverted repeat with a poly-T tract from higher plant chloroplast contributes to transcription termination of the translation-associated rps18 gene in a manner with the intrinsic termination, probably resulting from a heritage of endosymbiosis.

Full Text Available Abstract RNA synthesis and decay counteract each other and therefore inversely regulate gene expression in pro- and eukaryotic cells by controlling the steady-state level of individual transcripts. Genetic and biochemical data together with recent in depth annotation of bacterial genomes indicate that many components of the bacterial RNA decay machinery are evolutionarilyconserved and that their functional analogues exist in organisms belonging to all kingdoms of life. Here we briefly review biological functions of essential enzymes, their evolutionary conservation and multienzyme complexes that are involved in mRNA decay in Escherichia coli and discuss their conservation in evolutionarily distant bacteria.

Abstract Ecological genomics aims to understand the functional association between environmental gradients and the genes underlying adaptive traits. Many genes that are identified by genome-wide screening in ecologically relevant species lack functional annotations. Although gene functions can be inferred from sequence homology, such approaches have limited power. Here, we introduce ecological regulatory genomics by presenting an ontology-free gene prioritization method. Specifically, our method combines transcriptome profiling with high-throughput cis-regulatory sequence analysis in the water fleas Daphnia pulex and Daphnia magna. It screens coexpressed genes for overrepresented DNA motifs that serve as transcription factor binding sites, thereby providing insight into conservedtranscription factors and gene regulatory networks shaping the expression profile. We first validated our method, called Daphnia-cisTarget, on a D. pulex heat shock data set, which revealed a network driven by the heat shock factor. Next, we performed RNA-Seq in D. magna exposed to the cyanobacterium Microcystis aeruginosa. Daphnia-cisTarget identified coregulated gene networks that associate with the moulting cycle and potentially regulate life history changes in growth rate and age at maturity. These networks are predicted to be regulated by evolutionary conservedtranscription factors such as the homologues of Drosophila Shavenbaby and Grainyhead, nuclear receptors, and a GATA family member. In conclusion, our approach allows prioritising candidate genes in Daphnia without bias towards prior knowledge about functional gene annotation and represents an important step towards exploring the molecular mechanisms of ecological responses in organisms with poorly annotated genomes. PMID:28854641

Transcriptional regulation plays an important role in establishing gene expression profiles during development or in response to (a)biotic stimuli. Transcription factor binding sites (TFBSs) are the functional elements that determine transcriptional activity, and the identification of individual TFBS in genome sequences is a major goal to inferring regulatory networks. We have developed a phylogenetic footprinting approach for the identification of conserved noncoding sequences (CNSs) across 12 dicot plants. Whereas both alignment and non-alignment-based techniques were applied to identify functional motifs in a multispecies context, our method accounts for incomplete motif conservation as well as high sequence divergence between related species. We identified 69,361 footprints associated with 17,895 genes. Through the integration of known TFBS obtained from the literature and experimental studies, we used the CNSs to compile a gene regulatory network in Arabidopsis thaliana containing 40,758 interactions, of which two-thirds act through binding events located in DNase I hypersensitive sites. This network shows significant enrichment toward in vivo targets of known regulators, and its overall quality was confirmed using five different biological validation metrics. Finally, through the integration of detailed expression and function information, we demonstrate how static CNSs can be converted into condition-dependent regulatory networks, offering opportunities for regulatory gene annotation.

Planktonic microbial communities in the ocean are typically dominated by several cosmopolitan clades of Bacteria, Archaea, and Eukarya characterized by their ribosomal RNA gene phylogenies and genomic features. Although the environments these communities inhabit range from coastal to open ocean waters, how the biological dynamics vary between such disparate habitats is not well known. To gain insight into the differential activities of microbial populations inhabiting different oceanic provinces we compared the daily metatranscriptome profiles of related microbial populations inhabiting surface waters of both a coastal California upwelling region (CC) as well as the oligotrophic North Pacific Subtropical Gyre (NPSG). Transcriptional networks revealed that the dominant photoautotrophic microbes in each environment (Ostreococcus in CC, Prochlorococcus in NPSG) were central determinants of overall community transcriptome dynamics. Furthermore, heterotrophic bacterial clades common to both ecosystems (SAR11, SAR116, SAR86, SAR406, and Roseobacter) displayed conserved, genome-wide inter- and intrataxon transcriptional patterns and diel cycles. Populations of SAR11 and SAR86 clades in particular exhibited tightly coordinated transcriptional patterns in both coastal and pelagic ecosystems, suggesting that specific biological interactions between these groups are widespread in nature. Our results identify common diurnally oscillating behaviors among diverse planktonic microbial species regardless of habitat, suggesting that highly conserved temporally phased biotic interactions are ubiquitous among planktonic microbial communities worldwide.

A challenge in identifying the transcription regulatory region is that the locations of eukaryotic transcriptional elements are often diverse among different genes.SCN1A,a disease-related sodium channel gene,has a complex 5'-untranslated region and diverse mRNA transcripts,which might be driven by different promoters.By cross-species sequence comparison and bioinformatics analysis,human 5'-untranslated exons were found to be conserved within the region of 200 kb upstream of the 5' flanking regions of SCN1A in higher mammals,but not in lower mammals and non-mammals.The core promoter elements (INR,DPE,and TATA) were found in the regions flanking different 5'-untranslated exons,suggesting that these sequences (from-45 to+35) might be targeted as core promoters.The nucleotide identity rate of these core promoter sequences are different,and the conservation level of the upstream region of each core promoter varies distinctly,implicating different regulatory mechanisms of the four promoters which exist in the nervous system.

MicroRNAs (miRNAs) are endogenous 22-nt RNAs, which play important regulatory roles by post-transcriptional gene silencing. A computational strategy has been developed for the identification of conserved miRNAs based on features of known metazoan miRNAs in red flour beetle (Tribolium castaneum), which is regarded as one of the major laboratory models of arthropods. Among 118 putative miRNAs, 47% and 53% of the predicted miRNAs from the red flour beetle are harbored by known protein-coding genes (intronic) and genes located outside (intergenic miRNA), respectively. There are 31 intronic miRNAs in the same transcriptional orientation as the host genes, which may share RNA polymerase Ⅱ and spliceosomal machinery with their host genes for their biogenesis. A hypothetical feedback model has been proposed based on the analysis of the relationship between intronic miRNAs and their host genes in the development of red flour beetle.

Evolutionary conservation of DNA sequences provides a tool for the identification of functional elements in genomes. We have created a database of evolutionary conserved regions (ECRs) in vertebrate genomes entitled ECRbase that is constructed from a collection of pairwise vertebrate genome alignments produced by the ECR Browser database. ECRbase features a database of syntenic blocks that recapitulate the evolution of rearrangements in vertebrates and a collection of promoters in all vertebrate genomes presented in the database. The database also contains a collection of annotated transcription factor binding sites (TFBS) in all ECRs and promoter elements. ECRbase currently includes human, rhesus macaque, dog, opossum, rat, mouse, chicken, frog, zebrafish, and two pufferfish genomes. It is freely accessible at http://ECRbase.dcode.org.

Full Text Available Abstract Background Sox10, a member of the Sry-related HMG-Box gene family, is a critical transcription factor for several important cell lineages, most notably the neural crest stem cells and the derivative peripheral glial cells and melanocytes. Thus far, only a handful of direct target genes are known for this transcription factor limiting our understanding of the biological network it governs. Results We describe identification of multiple direct regulatory target genes of Sox10 through a procedure based on function and conservation. By combining RNA interference technique and DNA microarray technology, we have identified a set of genes that show significant down-regulation upon introduction of Sox10 specific siRNA into Schwannoma cells. Subsequent comparative genomics analyses led to potential binding sites for Sox10 protein conserved across several mammalian species within the genomic region proximal to these genes. Multiple sites belonging to 4 different genes (proteolipid protein, Sox10, extracellular superoxide dismutase, and pleiotrophin were shown to directly interact with Sox10 by chromatin immunoprecipitation assay. We further confirmed the direct regulation through the identified cis-element for one of the genes, extracellular superoxide dismutase, using electrophoretic mobility shift assay and reporter assay. Conclusion In sum, the process of combining differential expression profiling and comparative genomics successfully led to further defining the role of Sox10, a critical transcription factor for the development of peripheral glia. Our strategy utilizing relatively accessible techniques and tools should be applicable to studying the function of other transcription factors.

We introduce a method (MONKEY) to identify conservedtranscription-factor binding sites in multispecies alignments. MONKEY employs probabilistic models of factor specificity and binding site evolution, on which basis we compute the likelihood that putative sites are conserved and assign statistical significance to each hit. Using genomes from the genus Saccharomyces, we illustrate how the significance of real sites increases with evolutionary distance and explore the relationship between conservation and function.

We isolated several overlapping cDNA clones covering the 4242 nucleotides of a Strongylocentrotus purpuratus transcript that codes for a fibrillar procollagen chain. The sea urchin polypeptide includes a 124-amino acid long amino pre-propeptide, a 1064-amino acid alpha-chain inclusive of 338 uninterrupted Gly-X-Y repeats, and a 226-residue carboxyl-propeptide. The distribution of the highly conserved cysteines within the last domain together with the structural configuration of the amino-propeptide and the organization of the corresponding coding region, strongly suggest that the sea urchin gene is evolutionarily related to the vertebrate pro-alpha 2(I) collagen. This work, therefore, represents the first report of the complete primary structure of an invertebrate fibrillar procollagen chain. It also provides a new insight into the evolution of the amino-propeptide, the most divergent among the major protein domains of fibrillar procollagen chains.

Full Text Available The Fanconi anemia (FA gene family is a recent addition to the complex network of proteins that respond to and repair certain types of DNA damage in the human genome. Since little is known about the regulation of this novel group of genes at the DNA level, we characterized the promoters of the eight genes (FANCA, B, C, E, F, G, L and M that compose the FA core complex. The promoters of these genes show the characteristic attributes of housekeeping genes, such as a high GC content and CpG islands, a lack of TATA boxes and a low conservation. The promoters functioned in a monodirectional way and were, in their most active regions, comparable in strength to the SV40 promoter in our reporter plasmids. They were also marked by a distinctive transcriptional start site (TSS. In the 5' region of each promoter, we identified a region that was able to negatively regulate the promoter activity in HeLa and HEK 293 cells in isolation. The central and 3' regions of the promoter sequences harbor binding sites for several common and rare transcription factors, including STAT, SMAD, E2F, AP1 and YY1, which indicates that there may be cross-connections to several established regulatory pathways. Electrophoretic mobility shift assays and siRNA experiments confirmed the shared regulatory responses between the prominent members of the TGF-β and JAK/STAT pathways and members of the FA core complex. Although the promoters are not well conserved, they share region and sequence specific regulatory motifs and transcription factor binding sites (TBFs, and we identified a bi-partite nature to these promoters. These results support a hypothesis based on the co-evolution of the FA core complex genes that was expanded to include their promoters.

The glucose transporter 4 (GLUT4) plays a key role in glucose uptake in insulin target tissues. This transporter has been extensively studied in many species in terms of its function, expression and cellular traffic and complex mechanisms are involved in its regulation at many different levels. However, studies investigating the transcription of the GLUT4 gene and its regulation are scarce. In this study, we have identified the GLUT4 gene in a teleost fish, the Fugu (Takifugu rubripes), and have cloned and characterized a functional promoter of this gene for the first time in a non-mammalian vertebrate. In silico analysis of the Fugu GLUT4 promoter identified potential binding sites for transcription factors such as SP1, C/EBP, MEF2, KLF, SREBP-1c and GC-boxes, as well as a CpG island, but failed to identify a TATA box. In vitro analysis revealed three transcription start sites, with the main residing 307 bp upstream of the ATG codon. Deletion analysis determined that the core promoter was located between nucleotides -132/+94. By transfecting a variety of 5´deletion constructs into L6 muscle cells we have determined that Fugu GLUT4 promoter transcription is regulated by insulin, PG-J2, a PPARγ agonist, and electrical pulse stimulation. Furthermore, our results suggest the implication of motifs such as PPARγ/RXR and HIF-1α in the regulation of Fugu GLUT4 promoter activity by PPARγ and contractile activity, respectively. These data suggest that the characteristics and regulation of the GLUT4 promoter have been remarkably conserved during the evolution from fish to mammals, further evidencing the important role of GLUT4 in metabolic regulation in vertebrates.

Full Text Available To further understand the relationship between nucleosome-space occupancy (NO and global transcriptional activity in mammals, we acquired a set of genome-wide nucleosome distribution and transcriptome data from the mouse cerebrum and testis based on ChIP (H3-seq and RNA-seq, respectively. We identified a nearly consistent NO patterns among three mouse tissues--cerebrum, testis, and ESCs--and found, through clustering analysis for transcriptional activation, that the NO variations among chromosomes are closely associated with distinct expression levels between house-keeping (HK genes and tissue-specific (TS genes. Both TS and HK genes form clusters albeit the obvious majority. This feature implies that NO patterns, i.e. nucleosome binding and clustering, are coupled with gene clustering that may be functionally and evolutionarilyconserved in regulating gene expression among different cell types.

The Drosophila transcription factor Grainyhead regulates several key developmental processes. Three mammalian genes, CP2, LBP-1a and LBP-9 have been previously identified as homologues of grainyhead. We now report the cloning of two new mammalian genes (Mammalian grainyhead (MGR) and Brother-of-MGR (BOM)) and one new Drosophila gene (dCP2) that rewrite the phylogeny of this family. We demonstrate that MGR and BOM are more closely related to grh, whereas CP2, LBP-1a and LBP-9 are descendants of the dCP2 gene. MGR shares the greatest sequence homology with grh, is expressed in tissue-restricted patterns more comparable to grh and binds to and transactivates the promoter of the human Engrailed-1 gene, the mammalian homologue of the key grainyhead target gene, engrailed. This sequence and functional conservation indicates that the new mammalian members of this family play important developmental roles.

Borrelia burgdorferi BB0462 ORF encodes an unknown functional protein with 110 amino acids.A BLAST search in protein databases and the secondary structure being predicted by the program JUFO showed that the conserved hypothetical BB0462 protein was similar to the members of the YbaB protein family in both amino acid composition and protein structure.The co-transformation of BB0462 ORF and oppA upstream regulation DNA into E.coli host cells and β-galactosidase activity assay demonstrated that the BB0462 protein enhanced the transcriptional activity of the oppAV promoter,but does not affect those of oppAⅠ,Ⅱ,Ⅲ and Ⅳ promoters.Analysis of DNA retardation and competitive repression also confirmed that the BB0462 protein bound to the 409 bp upstream regulation DNA fragment close to the initiation codon of the oppAV gene.All data in our study suggested that the BB0462 protein was involved in the transcriptional regulation of the oppAV gene

Cells are often exposed to physical or chemical stresses that can damage the structures of essential biomolecules. Stress-induced cellular damage can become deleterious if not managed appropriately. Rapid and adaptive responses to stresses are therefore crucial for cell survival. In eukaryotic cells, different stresses trigger post-translational modification of proteins with the small ubiquitin-like modifier SUMO. However, the specific regulatory roles of sumoylation in each stress response are not well understood. Here, we examined the sumoylation events that occur in budding yeast after exposure to hyperosmotic stress. We discovered by proteomic and biochemical analyses that hyperosmotic stress incurs the rapid and transient sumoylation of Cyc8 and Tup1, which together form a conservedtranscription corepressor complex that regulates hundreds of genes. Gene expression and cell biological analyses revealed that sumoylation of each protein directs distinct outcomes. In particular, we discovered that Cyc8 sumoylation prevents the persistence of hyperosmotic stress-induced Cyc8-Tup1 inclusions, which involves a glutamine-rich prion domain in Cyc8. We propose that sumoylation protects against persistent inclusion formation during hyperosmotic stress, allowing optimal transcriptional function of the Cyc8-Tup1 complex. PMID:26800527

Objective Individuals with schizophrenia exhibit disturbances in a number of cognitive, affective, sensory, and motor functions that depend on the circuitry of different cortical areas. The cognitive deficits associated with dysfunction of the dorsolateral prefrontal cortex result, at least in part, from abnormalities in GABA neurotransmission, as reflected in a specific pattern of altered expression of GABA-related genes. Consequently, the authors sought to determine whether this pattern of altered gene expression is restricted to the dorsolateral prefrontal cortex or could also contribute to the dysfunction of other cortical areas in subjects with schizophrenia. Method Real-time quantitative polymerase chain reaction was used to assess the levels of eight GABA-related transcripts in four cortical areas (dorsolateral prefrontal cortex, anterior cingulate cortex, and primary motor and primary visual cortices) of subjects (N=12) with schizophrenia and matched normal comparison subjects. Results Expression levels of seven transcripts were lower in subjects with schizophrenia, with the magnitude of reduction for each transcript comparable across the four areas. The largest reductions were detected for mRNA encoding somatostatin and parvalbumin, followed by moderate decreases in mRNA expression for the 67-kilodalton isoform of glutamic acid decarboxylase, the GABA membrane transporter GAT-1, and the α1 and δ subunits of GABAA receptors. In contrast, the expression of calretinin mRNA did not differ between the subject groups in any of the four areas. Conclusions Because the areas examined represent the major functional domains (e.g., association, limbic, motor, and sensory) of the cerebral cortex, our findings suggest that a conserved set of molecular alterations affecting GABA neurotransmission contribute to the pathophysiology of different clinical features of schizophrenia. PMID:18281411

Full Text Available Non-coding DNA conservation across species has been often used as a predictor for transcriptional enhancer activity. However, only a few systematic analyses of the function of these highly conserved non-coding regions (HCNRs have been performed. Here we use zebrafish transgenic assays to perform a systematic study of 113 HCNRs from human chromosome 16. By comparing transient and stable transgenesis, we show that the first method is highly inefficient, leading to 40% of false positives and 20% of false negatives. When analyzed in stable transgenic lines, a great majority of HCNRs were active in the central nervous system, although some of them drove expression in other organs such as the eye and the excretory system. Finally, by testing a fraction of the HCNRs lacking enhancer activity for in vivo insulator activity, we find that 20% of them may contain enhancer-blocking function. Altogether our data indicate that HCNRs may contain different types of cis-regulatory activity, including enhancer, insulators as well as other not yet discovered functions.

The LSF/Grainyhead transcription factor family is involved in many important biological processes, including cell cycle, cell growth and development. In order to investigate the evolutionary conservation of these biological roles, we have characterized two new family members in Caenorhabditis elegans and Xenopus laevis. The C.elegans member, Ce-GRH-1, groups with the Grainyhead subfamily, while the X.laevis member, Xl-LSF, groups with the LSF subfamily. Ce-GRH-1 binds DNA in a sequence-specific manner identical to that of Drosophila melanogaster Grainyhead. In addition, Ce-GRH-1 binds to sequences upstream of the C.elegans gene encoding aromatic L-amino-acid decarboxylase and genes involved in post-embryonic development, mab-5 and dbl-1. All three C.elegans genes are homologs of D.melanogaster Grainyhead-regulated genes. RNA-mediated interference of Ce-grh-1 results in embryonic lethality in worms, accompanied by soft, defective cuticles. These phenotypes are strikingly similar to those observed previously in D.melanogaster grainyhead mutants, suggesting conservation of the developmental role of these family members over the course of evolution. Our phylogenetic analysis of the expanded LSF/GRH family (including other previously unrecognized proteins/ESTs) suggests that the structural and functional dichotomy of this family dates back more than 700 million years, i.e. to the time when the first multicellular organisms are thought to have arisen.

Full Text Available The elongation phase of transcription by RNA Polymerase II (Pol II involves numerous events that are tightly coordinated, including RNA processing, histone modification, and chromatin remodeling. RNA splicing factors are associated with elongating Pol II, and the interdependent coupling of splicing and elongation has been documented in several systems. Here we identify a conserved, multi-domain cyclophilin family member, SIG-7, as an essential factor for both normal transcription elongation and co-transcriptional splicing. In embryos depleted for SIG-7, RNA levels for over a thousand zygotically expressed genes are substantially reduced, Pol II becomes significantly reduced at the 3' end of genes, marks of transcription elongation are reduced, and unspliced mRNAs accumulate. Our findings suggest that SIG-7 plays a central role in both Pol II elongation and co-transcriptional splicing and may provide an important link for their coordination and regulation.

The elongation phase of transcription by RNA Polymerase II (Pol II) involves numerous events that are tightly coordinated, including RNA processing, histone modification, and chromatin remodeling. RNA splicing factors are associated with elongating Pol II, and the interdependent coupling of splicing and elongation has been documented in several systems. Here we identify a conserved, multi-domain cyclophilin family member, SIG-7, as an essential factor for both normal transcription elongation and co-transcriptional splicing. In embryos depleted for SIG-7, RNA levels for over a thousand zygotically expressed genes are substantially reduced, Pol II becomes significantly reduced at the 3’ end of genes, marks of transcription elongation are reduced, and unspliced mRNAs accumulate. Our findings suggest that SIG-7 plays a central role in both Pol II elongation and co-transcriptional splicing and may provide an important link for their coordination and regulation. PMID:27541139

Thioredoxins are low molecular weight redox proteins, alternating between the S-S (oxidized) and SH (reduced) states, that function in a number of biochemical processes, including DNA synthesis, DNA replication, and enzyme regulation. Until recently, reduced ferredoxin was known to serve as the source of reducing power for the reduction of thioredoxins only in oxygenic photosynthetic cells. In all other organisms, the source of hydrogen (electrons) for thioredoxin reduction was considered to be NADPH. It was found that Clostridium pasteurianum, an anaerobic organism normally living in the soil unexposed to light, resembles photosynthetic cells in using ferredoxin for the reduction of thioredoxin. The results reveal the existence of a pathway in which ferredoxin, provides the reducing power for the reduction of thioredoxin via the flavoprotein enzyme, ferredoxinthioredoxin reductase. In related studies, it was found that Chromatium vinosum, an anaerobic photosynthetic purple sulfur bacterium, resembles evolutionarily more advanced micro-organisms in having an NADP-thioredoxin system composed of a single thioredoxin which is reduced by NADPH via NADP-thioredoxin reductase. The adoption of the NADP-thioredoxin system by Chromatium seems appropriate in view of evidence tha the organi sm utilizes ATP-driven reverse electron transport. Finally, results of research directed towards the identification of target enzymes of the ferredoxin/thioredoxin system in a cyanobacterium (Nostoc muscorum), show that thioredoxin-linked photosynthetic enzymes of cyanobateria are similar to those of chloroplasts. It now seems that the ferredoxin/thioredoxin system functions in regulating CO2 assimilation via the reductive pentose phosphate cycle in oxygenic but not anoxygenic photosynthetic cells.

Prediction of transcription-factor target sites in promoters remains difficult due to the short length and degeneracy of the target sequences. Although the use of orthologous sequences and phylogenetic footprinting approaches may help in the recognition of conserved and potentially functional sequen

How extracellular cues are transduced to the nucleus is a fundamental issue in biology. The paralogous WW-domain proteins YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif; also known as WWTR1, for WW-domain containing transcription regulator 1) constitute a pair of transducers linking cytoplasmic signaling events to transcriptional regulation in the nucleus. A cascade composed of mammalian Ste20-like (MST) and large tumor suppressor (LATS) kinases directs multisite phosphorylation, promotes 14-3-3 binding, and hinders nuclear import of YAP and TAZ, thereby inhibiting their transcriptional coactivator and growth-promoting activities. A similar cascade regulates the trafficking and function of Yorkie, the fly orthologue of YAP. Mammalian YAP and TAZ are expressed in various tissues and serve as coregulators for transcriptional enhancer factors (TEFs; also referred to as TEADs, for TEA-domain proteins), runt-domain transcription factors (Runxs), peroxisome proliferator-activated receptor gamma (PPARgamma), T-box transcription factor 5 (Tbx5), and several others. YAP and TAZ play distinct roles during mouse development. Both, and their upstream regulators, are intimately linked to tumorigenesis and other pathogenic processes. Here, we review studies on this family of signal-responsive transcriptional coregulators and emphasize how relative sequence conservation predicates their function and regulation, to provide a conceptual framework for organizing available information and seeking new knowledge about these signal transducers.

The mraZ and mraW genes are highly conserved in bacteria, both in sequence and location at the head of the division and cell wall (dcw) gene cluster. Although MraZ has structural similarity to the AbrB transition state regulator and the MazE antitoxin, and MraW is known to methylate ribosomal RNA, mraZ and mraW null mutants have no detectable growth phenotype in any species tested to date, hampering progress in understanding their physiological role. Here we show that overproduction of Escherichia coli MraZ perturbs cell division and the cell envelope, is more lethal at high levels or in minimal growth medium, and that MraW antagonizes these effects. MraZGFP localizes to the nucleoid, suggesting that it binds DNA. Indeed, purified MraZ directly binds a region upstream from its own promoter containing three direct repeats to regulate its own expression and that of downstream cell division and cell wall genes. MraZ-LacZ fusions are repressed by excess MraZ but not when DNA binding by MraZ is inhibited. RNAseq analysis indicates that MraZ is a global transcriptional regulator with numerous targets in addition to dcw genes. One of these targets, mioC, is directly bound by MraZ in a region with three direct repeats.

BK polyomavirus (BKV) is widespread among humans, asymptomatically infecting children and then persisting in renal tissue. The transcriptional control region (TCR) of the BKV genome is variable among clinical isolates. Thus, archetypal TCRs with a common basic configuration generally occur in BKV isolates from the urine of immunocompromised patients, but rearranged TCRs that possibly arise from the archetypal configuration have also been detected in clinical specimens. To examine the hypothesis that archetypal strains represent wild-type strains circulating in the human population (the archetype hypothesis), we analysed 145 complete viral genomes amplified directly from the urine of non-immunocompromised individuals worldwide. These genomes included 82, three, two and 58 sequences classified as belonging to subtypes I, II, III and IV, respectively. Rearranged TCRs with long duplications or deletions were detected from two subtype I and two subtype IV genomes, but not from the other 141 genomes (thus, the TCRs of these genomes were judged to be archetypal). The variations in the archetypal TCRs were nucleotide substitutions and single-nucleotide deletions, most of which were unique to particular subtypes or subgroups. We confirmed that the four complete BKV genomes with rearranged TCRs did not form a unique lineage on a phylogenetic tree. Collectively, the findings demonstrate that the archetypal TCR configuration has been conserved during the evolution of BKV, providing support for the archetype hypothesis. Additionally, we suggest that 'archetype' should be used as a conceptual term that denotes a prototypical structure that can generate various rearranged TCRs during viral growth in vivo and in vitro.

Errors that occur during transcription have received much less attention than the mutations that occur in DNA because transcription errors are not heritable and usually result in a very limited number of altered proteins. However, transcription error rates are typically several orders of magnitude higher than the mutation rate. Also, individual transcripts can be translated multiple times, so a single error can have substantial effects on the pool of proteins. Transcription errors can also contribute to cellular noise, thereby influencing cell survival under stressful conditions, such as starvation or antibiotic stress. Implementing a method that captures transcription errors genome-wide, we measured the rates and spectra of transcription errors in Escherichia coli and in endosymbionts for which mutation and/or substitution rates are greatly elevated over those of E. coli. Under all tested conditions, across all species, and even for different categories of RNA sequences (mRNA and rRNAs), there were no significant differences in rates of transcription errors, which ranged from 2.3 × 10−5 per nucleotide in mRNA of the endosymbiont Buchnera aphidicola to 5.2 × 10−5 per nucleotide in rRNA of the endosymbiont Carsonella ruddii. The similarity of transcription error rates in these bacterial endosymbionts to that in E. coli (4.63 × 10−5 per nucleotide) is all the more surprising given that genomic erosion has resulted in the loss of transcription fidelity factors in both Buchnera and Carsonella. PMID:26884158

Errors that occur during transcription have received much less attention than the mutations that occur in DNA because transcription errors are not heritable and usually result in a very limited number of altered proteins. However, transcription error rates are typically several orders of magnitude higher than the mutation rate. Also, individual transcripts can be translated multiple times, so a single error can have substantial effects on the pool of proteins. Transcription errors can also contribute to cellular noise, thereby influencing cell survival under stressful conditions, such as starvation or antibiotic stress. Implementing a method that captures transcription errors genome-wide, we measured the rates and spectra of transcription errors in Escherichia coli and in endosymbionts for which mutation and/or substitution rates are greatly elevated over those of E. coli Under all tested conditions, across all species, and even for different categories of RNA sequences (mRNA and rRNAs), there were no significant differences in rates of transcription errors, which ranged from 2.3 × 10(-5) per nucleotide in mRNA of the endosymbiont Buchnera aphidicola to 5.2 × 10(-5) per nucleotide in rRNA of the endosymbiont Carsonella ruddii The similarity of transcription error rates in these bacterial endosymbionts to that in E. coli (4.63 × 10(-5) per nucleotide) is all the more surprising given that genomic erosion has resulted in the loss of transcription fidelity factors in both Buchnera and Carsonella.

Full Text Available Cytochrome P450 monooxygenases (P450s are heme-thiolate proteins distributed across the biological kingdoms. P450s are catalytically versatile and play key roles in organisms primary and secondary metabolism. Identification of P450s across the biological kingdoms depends largely on the identification of two P450 signature motifs, EXXR and CXG, in the protein sequence. Once a putative protein has been identified as P450, it will be assigned to a family and subfamily based on the criteria that P450s within a family share more than 40% homology and members of subfamilies share more than 55% homology. However, to date, no evidence has been presented that can distinguish members of a P450 family. Here, for the first time we report the identification of EXXR- and CXG-motifs-based amino acid patterns that are characteristic of the P450 family. Analysis of P450 signature motifs in the under-explored fungal P450s from four different phyla, ascomycota, basidiomycota, zygomycota and chytridiomycota, indicated that the EXXR motif is highly variable and the CXG motif is somewhat variable. The amino acids threonine and leucine are preferred as second and third amino acids in the EXXR motif and proline and glycine are preferred as second and third amino acids in the CXG motif in fungal P450s. Analysis of 67 P450 families from biological kingdoms such as plants, animals, bacteria and fungi showed conservation of a set of amino acid patterns characteristic of a particular P450 family in EXXR and CXG motifs. This suggests that during the divergence of P450 families from a common ancestor these amino acids patterns evolve and are retained in each P450 family as a signature of that family. The role of amino acid patterns characteristic of a P450 family in the structural and/or functional aspects of members of the P450 family is a topic for future research.

Full Text Available Abstract Background The Solanaceae is a family of closely related species with diverse phenotypes that have been exploited for agronomic purposes. Previous studies involving a small number of genes suggested sequence conservation across the Solanaceae. The availability of large collections of Expressed Sequence Tags (ESTs for the Solanaceae now provides the opportunity to assess sequence conservation and divergence on a genomic scale. Results All available ESTs and Expressed Transcripts (ETs, 449,224 sequences for six Solanaceae species (potato, tomato, pepper, petunia, tobacco and Nicotiana benthamiana, were clustered and assembled into gene indices. Examination of gene ontologies revealed that the transcripts within the gene indices encode a similar suite of biological processes. Although the ESTs and ETs were derived from a variety of tissues, 55–81% of the sequences had significant similarity at the nucleotide level with sequences among the six species. Putative orthologs could be identified for 28–58% of the sequences. This high degree of sequence conservation was supported by expression profiling using heterologous hybridizations to potato cDNA arrays that showed similar expression patterns in mature leaves for all six solanaceous species. 16–19% of the transcripts within the six Solanaceae gene indices did not have matches among Solanaceae, Arabidopsis, rice or 21 other plant gene indices. Conclusion Results from this genome scale analysis confirmed a high level of sequence conservation at the nucleotide level of the coding sequence among Solanaceae. Additionally, the results indicated that part of the Solanaceae transcriptome is likely to be unique for each species.

The majority of the eukaryotic genome is transcribed, generating a significant number of long intergenic noncoding RNAs (lincRNAs). Although lincRNAs represent the most poorly understood product of transcription, recent work has shown lincRNAs fulfill important cellular functions. In addition to low sequence conservation, poor understanding of structural mechanisms driving lincRNA biology hinders systematic prediction of their function. Here we report the molecular requirements for the recognition of steroid receptors (SRs) by the lincRNA growth arrest-specific 5 (Gas5), which regulates steroid-mediated transcriptional regulation, growth arrest and apoptosis. We identify the functional Gas5-SR interface and generate point mutations that ablate the SR-Gas5 lincRNA interaction, altering Gas5-driven apoptosis in cancer cell lines. Further, we find that the Gas5 SR-recognition sequence is conserved among haplorhines, with its evolutionary origin as a splice acceptor site. This study demonstrates that lincRNAs can recognize protein targets in a conserved, sequence-specific manner in order to affect critical cell functions.

Full Text Available In animals, replication-independent incorporation of nucleosomes containing the histone variant H3.3 enables global reprogramming of histone modifications and transcriptional profiles. H3.3 enrichment over gene bodies correlates with gene transcription in animals and plants. In animals, H3.3 is deposited into chromatin by specific protein complexes, including the HIRA complex. H3.3 variants evolved independently and acquired similar properties in animals and plants, questioning how the H3.3 deposition machinery evolved in plants and what are its biological functions. We performed phylogenetic analyses in the plant kingdom and identified in Arabidopsis all orthologs of human genes encoding members of the HIRA complex. Genetic analyses, biochemical data and protein localisation suggest that these proteins form a complex able to interact with H3.3 in Arabidopsis in a manner similar to that described in mammals. In contrast to animals, where HIRA is required for fertilization and early development, loss of function of HIRA in Arabidopsis causes mild phenotypes in the adult plant and does not perturb sexual reproduction and embryogenesis. Rather, HIRA function is required for transcriptional reprogramming during dedifferentiation of plant cells that precedes vegetative propagation and for the appropriate transcription of genes responsive to biotic and abiotic factors. We conclude that the molecular function of the HIRA complex is conserved between plants and animals. Yet plants diversified HIRA functions to enable asexual reproduction and responsiveness to the environment in response to the plant sessile lifestyle.

Background The identification of transcription factor binding sites is difficult since they are only a small number of nucleotides in size, resulting in large numbers of false positives and false negatives in current approaches. Computational methods to reduce false positives are to look for over-representation of transcription factor binding sites in a set of similarly regulated promoters or to look for conservation in orthologous promoter alignments. Results We have developed a novel tool, "CORE_TF" (Conserved and Over-REpresented Transcription Factor binding sites) that identifies common transcription factor binding sites in promoters of co-regulated genes. To improve upon existing binding site predictions, the tool searches for position weight matrices from the TRANSFACR database that are over-represented in an experimental set compared to a random set of promoters and identifies cross-species conservation of the predicted transcription factor binding sites. The algorithm has been evaluated with expression and chromatin-immunoprecipitation on microarray data. We also implement and demonstrate the importance of matching the random set of promoters to the experimental promoters by GC content, which is a unique feature of our tool. Conclusion The program CORE_TF is accessible in a user friendly web interface at . It provides a table of over-represented transcription factor binding sites in the users input genes' promoters and a graphical view of evolutionary conservedtranscription factor binding sites. In our test data sets it successfully predicts target transcription factors and their binding sites. PMID:19036135

Animal development is complex yet surprisingly robust. Animals may develop alternative phenotypes conditional on environmental changes. Under unfavorable conditions, Caenorhabditis elegans larvae enter the dauer stage, a developmentally arrested, long-lived, and stress-resistant state. Dauer larvae of free-living nematodes and infective larvae of parasitic nematodes share many traits including a conserved endocrine signaling module (DA/DAF-12), which is essential for the formation of dauer and infective larvae. We speculated that conserved post-transcriptional regulatory mechanism might also be involved in executing the dauer and infective larvae fate. We used an unbiased sequencing strategy to characterize the microRNA (miRNA) gene complement in C. elegans, Pristionchus pacificus, and Strongyloides ratti. Our study raised the number of described miRNA genes to 257 for C. elegans, tripled the known gene set for P. pacificus to 362 miRNAs, and is the first to describe miRNAs in a Strongyloides parasite. Moreover, we found a limited core set of 24 conserved miRNA families in all three species. Interestingly, our estimated expression fold changes between dauer versus nondauer stages and infective larvae versus free-living stages reveal that despite the speed of miRNA gene set evolution in nematodes, homologous gene families with conserved "dauer-infective" expression signatures are present. These findings suggest that common post-transcriptional regulatory mechanisms are at work and that the same miRNA families play important roles in developmental arrest and long-term survival in free-living and parasitic nematodes.

Full Text Available Abstract Background Cis-regulatory modules (CRMs are distinct, genomic regions surrounding the target gene that can independently activate the promoter to drive transcription. The activation of a CRM is controlled by the binding of a certain combination of transcription factors (TFs. It would be of great benefit if the transcriptional output mediated by a specific CRM could be predicted. Of equal benefit would be identifying in silico a specific CRM as the driver of the expression in a specific tissue or situation. We extend a recently developed biochemical modeling approach to manage both prediction tasks. Given a set of TFs, their protein concentrations, and the positions and binding strengths of each of the TFs in a putative CRM, the model predicts the transcriptional output of the gene. Our approach predicts the location of the regulating CRM by using predicted TF binding sites in regions near the gene as input to the model and searching for the region that yields a predicted transcription rate most closely matching the known rate. Results Here we show the ability of the model on the example of one of the CRMs regulating the eve gene, MSE2. A model trained on the MSE2 in D. melanogaster was applied to the surrounding sequence of the eve gene in seven other Drosophila species. The model successfully predicts the correct MSE2 location and output in six out of eight Drosophila species we examine. Conclusion The model is able to generalize from D. melanogaster to other Drosophila species and accurately predicts the location and transcriptional output of MSE2 in those species. However, we also show that the current model is not specific enough to function as a genome-wide CRM scanner, because it incorrectly predicts other genomic regions to be MSE2s.

Cis-regulatory modules (CRMs) are distinct, genomic regions surrounding the target gene that can independently activate the promoter to drive transcription. The activation of a CRM is controlled by the binding of a certain combination of transcription factors (TFs). It would be of great benefit if the transcriptional output mediated by a specific CRM could be predicted. Of equal benefit would be identifying in silico a specific CRM as the driver of the expression in a specific tissue or situation. We extend a recently developed biochemical modeling approach to manage both prediction tasks. Given a set of TFs, their protein concentrations, and the positions and binding strengths of each of the TFs in a putative CRM, the model predicts the transcriptional output of the gene. Our approach predicts the location of the regulating CRM by using predicted TF binding sites in regions near the gene as input to the model and searching for the region that yields a predicted transcription rate most closely matching the known rate. Here we show the ability of the model on the example of one of the CRMs regulating the eve gene, MSE2. A model trained on the MSE2 in D. melanogaster was applied to the surrounding sequence of the eve gene in seven other Drosophila species. The model successfully predicts the correct MSE2 location and output in six out of eight Drosophila species we examine. The model is able to generalize from D. melanogaster to other Drosophila species and accurately predicts the location and transcriptional output of MSE2 in those species. However, we also show that the current model is not specific enough to function as a genome-wide CRM scanner, because it incorrectly predicts other genomic regions to be MSE2s.

Transcript patterns elicited in response to attack reveal, at the molecular level, how plants respond to aggressors. These patterns are fashioned both by inflicted physical damage as well as by biological components displayed or released by the attacker. Different types of attacking organisms might

Escherichia coli heat shock transcription factor sigma32 is rapidly degraded in vivo, with a half-life of about 1 min. A set of proteins that includes the DnaK chaperone team (DnaK, DnaJ, GrpE) and ATP-dependent proteases (FtsH, HslUV, etc.) are involved in degradation of sigma32. To gain further insight into the regulation of sigma32 stability, we isolated sigma32 mutants that were markedly stabilized. Many of the mutants had amino acid substitutions in the N-terminal half (residues 47 to 55) of region 2.1, a region highly conserved among bacterial sigma factors. The half-lives ranged from about 2-fold to more than 10-fold longer than that of the wild-type protein. Besides greater stability, the levels of heat shock proteins, such as DnaK and GroEL, increased in cells producing stable sigma32. Detailed analysis showed that some stable sigma32 mutants have higher transcriptional activity than the wild type. These results indicate that the N-terminal half of region 2.1 is required for modulating both metabolic stability and the activity of sigma32. The evidence suggests that sigma32 stabilization does not result from an elevated affinity for core RNA polymerase. Region 2.1 may, therefore, be involved in interactions with the proteolytic machinery, including molecular chaperones.

Innate immunity in Caenorhabditis elegans requires a conserved PMK-1 p38 mitogen-activated protein kinase (MAPK) pathway that regulates the basal and pathogen-induced expression of immune effectors. The mechanisms by which PMK-1 p38 MAPK regulates the transcriptional activation of the C. elegans immune response have not been identified. Furthermore, in mammalian systems the genetic analysis of physiological targets of p38 MAPK in immunity has been limited. Here, we show that C. elegans ATF-7, a member of the conserved cyclic AMP-responsive element binding (CREB)/activating transcription factor (ATF) family of basic-region leucine zipper (bZIP) transcription factors and an ortholog of mammalian ATF2/ATF7, has a pivotal role in the regulation of PMK-1-mediated innate immunity. Genetic analysis of loss-of-function alleles and a gain-of-function allele of atf-7, combined with expression analysis of PMK-1-regulated genes and biochemical characterization of the interaction between ATF-7 and PMK-1, suggest that ATF-7 functions as a repressor of PMK-1-regulated genes that undergoes a switch to an activator upon phosphorylation by PMK-1. Whereas loss-of-function mutations in atf-7 can restore basal expression of PMK-1-regulated genes observed in the pmk-1 null mutant, the induction of PMK-1-regulated genes by pathogenic Pseudomonas aeruginosa PA14 is abrogated. The switching modes of ATF-7 activity, from repressor to activator in response to activated PMK-1 p38 MAPK, are reminiscent of the mechanism of regulation mediated by the corresponding ancestral Sko1p and Hog1p proteins in the yeast response to osmotic stress. Our data point to the regulation of the ATF2/ATF7/CREB5 family of transcriptional regulators by p38 MAPK as an ancient conserved mechanism for the control of innate immunity in metazoans, and suggest that ATF2/ATF7 may function in a similar manner in the regulation of mammalian innate immunity.

Full Text Available Innate immunity in Caenorhabditis elegans requires a conserved PMK-1 p38 mitogen-activated protein kinase (MAPK pathway that regulates the basal and pathogen-induced expression of immune effectors. The mechanisms by which PMK-1 p38 MAPK regulates the transcriptional activation of the C. elegans immune response have not been identified. Furthermore, in mammalian systems the genetic analysis of physiological targets of p38 MAPK in immunity has been limited. Here, we show that C. elegans ATF-7, a member of the conserved cyclic AMP-responsive element binding (CREB/activating transcription factor (ATF family of basic-region leucine zipper (bZIP transcription factors and an ortholog of mammalian ATF2/ATF7, has a pivotal role in the regulation of PMK-1-mediated innate immunity. Genetic analysis of loss-of-function alleles and a gain-of-function allele of atf-7, combined with expression analysis of PMK-1-regulated genes and biochemical characterization of the interaction between ATF-7 and PMK-1, suggest that ATF-7 functions as a repressor of PMK-1-regulated genes that undergoes a switch to an activator upon phosphorylation by PMK-1. Whereas loss-of-function mutations in atf-7 can restore basal expression of PMK-1-regulated genes observed in the pmk-1 null mutant, the induction of PMK-1-regulated genes by pathogenic Pseudomonas aeruginosa PA14 is abrogated. The switching modes of ATF-7 activity, from repressor to activator in response to activated PMK-1 p38 MAPK, are reminiscent of the mechanism of regulation mediated by the corresponding ancestral Sko1p and Hog1p proteins in the yeast response to osmotic stress. Our data point to the regulation of the ATF2/ATF7/CREB5 family of transcriptional regulators by p38 MAPK as an ancient conserved mechanism for the control of innate immunity in metazoans, and suggest that ATF2/ATF7 may function in a similar manner in the regulation of mammalian innate immunity.

Full Text Available The Giardia lamblia cyst wall is required for survival outside the host and infection. Three cyst wall protein (cwp genes identified to date are highly up-regulated during encystation. However, little is known of the molecular mechanisms governing their gene regulation. Messenger RNAs containing premature stop codons are rapidly degraded by a nonsense-mediated mRNA decay (NMD system to avoid production of non-functional proteins. In addition to RNA surveillance, NMD also regulates thousands of naturally occurring transcripts through a variety of mechanisms. It is interesting to know the NMD pathway in the primitive eukaryotes. Previously, we have found that the giardial homologue of a conserved NMD factor, UPF1, may be functionally conserved and involved in NMD and in preventing nonsense suppression. In this study, we tested the hypothesis that NMD factors can regulate some naturally occurring transcripts in G. lamblia. We found that overexpression of UPF1 resulted in a significant decrease of the levels of CWP1 and cyst formation and of the endogenous cwp1-3, and myb2 mRNA levels and stability. This indicates that NMD could contribute to the regulation of the cwp1-3 and myb2 transcripts, which are key to G. lamblia differentiation into cyst. Interestingly, we also found that UPF1 may be involved in regulation of eight other endogenous genes, including up-regulation of the translation elongation factor gene, whose product increases translation which is required for NMD. Our results indicate that NMD factor could contribute to the regulation of not only nonsense containing mRNAs, but also mRNAs of the key encystation-induced genes and other endogenous genes in the early-diverging eukaryote, G. lamblia.

Full Text Available Transcription coupled nucleotide excision repair (TC-NER is involved in correcting UV-induced damage and other road-blocks encountered in the transcribed strand. Mutation frequency decline (Mfd is a transcription repair coupling factor, involved in repair of template strand during transcription. Mfd from M. tuberculosis (MtbMfd is 1234 amino-acids long harboring characteristic modules for different activities. Mtbmfd complemented Escherichia coli mfd (Ecomfd deficient strain, enhanced survival of UV irradiated cells and increased the road-block repression in vivo. The protein exhibited ATPase activity, which was stimulated ∼1.5-fold in the presence of DNA. While the C-terminal domain (CTD comprising amino acids 630 to 1234 showed ∼2-fold elevated ATPase activity than MtbMfd, the N-terminal domain (NTD containing the first 433 amino acid residues was able to bind ATP but deficient in hydrolysis. Overexpression of NTD of MtbMfd led to growth defect and hypersensitivity to UV light. Deletion of 184 amino acids from the C-terminal end of MtbMfd (MfdΔC increased the ATPase activity by ∼10-fold and correspondingly exhibited efficient translocation along DNA as compared to the MtbMfd and CTD. Surprisingly, MtbMfd was found to be distributed in monomer and hexamer forms both in vivo and in vitro and the monomer showed increased susceptibility to proteases compared to the hexamer. MfdΔC, on the other hand, was predominantly monomeric in solution implicating the extreme C-terminal region in oligomerization of the protein. Thus, although the MtbMfd resembles EcoMfd in many of its reaction characteristics, some of its hitherto unknown distinct properties hint at its species specific role in mycobacteria during transcription-coupled repair.

Full Text Available The Nav1.7 voltage-gated sodium channel, encoded by SCN9A, is critical for human pain perception yet the transcriptional and post-transcriptional mechanisms that regulate this gene are still incompletely understood. Here, we describe a novel natural antisense transcript (NAT for SCN9A that is conserved in humans and mice. The NAT has a similar tissue expression pattern to the sense gene and is alternatively spliced within dorsal root ganglia. The human and mouse NATs exist in cis with the sense gene in a tail-to-tail orientation and both share sequences that are complementary to the terminal exon of SCN9A/Scn9a. Overexpression analyses of the human NAT in human embryonic kidney (HEK293A and human neuroblastoma (SH-SY5Y cell lines show that it can function to downregulate Nav1.7 mRNA, protein levels and currents. The NAT may play an important role in regulating human pain thresholds and is a potential candidate gene for individuals with chronic pain disorders that map to the SCN9A locus, such as Inherited Primary Erythromelalgia, Paroxysmal Extreme Pain Disorder and Painful Small Fibre Neuropathy, but who do not contain mutations in the sense gene. Our results strongly suggest the SCN9A NAT as a prime candidate for new therapies based upon augmentation of existing antisense RNAs in the treatment of chronic pain conditions in man.

The Nav1.7 voltage-gated sodium channel, encoded by SCN9A, is critical for human pain perception yet the transcriptional and post-transcriptional mechanisms that regulate this gene are still incompletely understood. Here, we describe a novel natural antisense transcript (NAT) for SCN9A that is conserved in humans and mice. The NAT has a similar tissue expression pattern to the sense gene and is alternatively spliced within dorsal root ganglia. The human and mouse NATs exist in cis with the sense gene in a tail-to-tail orientation and both share sequences that are complementary to the terminal exon of SCN9A/Scn9a. Overexpression analyses of the human NAT in human embryonic kidney (HEK293A) and human neuroblastoma (SH-SY5Y) cell lines show that it can function to downregulate Nav1.7 mRNA, protein levels and currents. The NAT may play an important role in regulating human pain thresholds and is a potential candidate gene for individuals with chronic pain disorders that map to the SCN9A locus, such as Inherited Primary Erythromelalgia, Paroxysmal Extreme Pain Disorder and Painful Small Fibre Neuropathy, but who do not contain mutations in the sense gene. Our results strongly suggest the SCN9A NAT as a prime candidate for new therapies based upon augmentation of existing antisense RNAs in the treatment of chronic pain conditions in man.

A general initiation factor, TFIIE, is essential for transcription initiation by RNA polymerase II in conjunction with other general factors. TFIIE is a heterotetramer containing two subunits of relative molecular mass 57,000 (TFIIE-alpha) and two of 34,000 (TFIIE-beta). TFIIE-beta is required in conjunction with TFIIE-alpha for transcription initiation. Here we report the cloning and expression of a complementary DNA encoding a functional human TFIIE-beta. Recombinant TFIIE-beta could replace the natural TFIIE-beta for transcription in conjunction with TFIIE-alpha. Amino-acid sequence comparisons reveal regions with sequence similarities to: subregion 3 of bacterial sigma factors; a region of RAP30 (the small subunit of TFIIF) with sequence similarity to a sigma-factor subregion implicated in binding to RNA polymerase; and a portion of the basic region-helix-loop-helix motif found in several enhancer-binding proteins. These potential homologies have implications for the role of TFIIE in preinitiation complex assembly and function.

Functional and anatomical sexual dimorphisms in the brain are either the result of cells that are generated only in one sex or a manifestation of sex-specific differentiation of neurons present in both sexes. The PHC neuron pair of the nematode C. elegans differentiates in a strikingly sex-specific manner. In hermaphrodites the PHC neurons display a canonical pattern of synaptic connectivity similar to that of other sensory neurons, but in males PHC differentiates into a densely connected hub sensory neuron/interneuron, integrating a large number of male-specific synaptic inputs and conveying them to both male-specific and sex-shared circuitry. We show that the differentiation into such a hub neuron involves the sex-specific scaling of several components of the synaptic vesicle machinery, including the vesicular glutamate transporter eat-4/VGLUT, induction of neuropeptide expression, changes in axonal projection morphology, and a switch in neuronal function. We demonstrate that these molecular and anatomical remodeling events are controlled cell autonomously by the phylogenetically conserved Doublesex homolog dmd-3, which is both required and sufficient for sex-specific PHC differentiation. Cellular specificity of dmd-3 action is ensured by its collaboration with non-sex-specific terminal selector-type transcription factors, whereas the sex specificity of dmd-3 action is ensured by the hermaphrodite-specific transcriptional master regulator of hermaphroditic cell identity tra-1, which represses the transcription of dmd-3 in hermaphrodite PHC. Taken together, our studies provide mechanistic insights into how neurons are specified in a sexually dimorphic manner.

Full Text Available Ultraconserved elements (UCEs have been the subject of great interest because of their extreme sequence identity and their seemingly cryptic and largely uncharacterized functions. Although in vivo studies of UCE sequences have demonstrated regulatory activity, protein interactors at UCEs have not been systematically identified. Here, we combined high-throughput affinity purification, high-resolution mass spectrometry, and SILAC quantification to map intrinsic protein interactions for 193 UCE sequences. The interactome contains over 400 proteins, including transcription factors with known developmental roles. We demonstrate based on our data that UCEs consist of strongly conserved overlapping binding sites. We also generated a fine-resolution interactome of a UCE, confirming the hub-like nature of the element. The intrinsic interactions mapped here are reflected in open chromatin, as indicated by comparison with existing ChIP data. Our study argues for a strong contribution of protein-DNA interactions to UCE conservation and provides a basis for further functional characterization of UCEs.

Full Text Available Abstract Background The identification of transcription factor binding sites is difficult since they are only a small number of nucleotides in size, resulting in large numbers of false positives and false negatives in current approaches. Computational methods to reduce false positives are to look for over-representation of transcription factor binding sites in a set of similarly regulated promoters or to look for conservation in orthologous promoter alignments. Results We have developed a novel tool, "CORE_TF" (Conserved and Over-REpresented Transcription Factor binding sites that identifies common transcription factor binding sites in promoters of co-regulated genes. To improve upon existing binding site predictions, the tool searches for position weight matrices from the TRANSFACR database that are over-represented in an experimental set compared to a random set of promoters and identifies cross-species conservation of the predicted transcription factor binding sites. The algorithm has been evaluated with expression and chromatin-immunoprecipitation on microarray data. We also implement and demonstrate the importance of matching the random set of promoters to the experimental promoters by GC content, which is a unique feature of our tool. Conclusion The program CORE_TF is accessible in a user friendly web interface at http://www.LGTC.nl/CORE_TF. It provides a table of over-represented transcription factor binding sites in the users input genes' promoters and a graphical view of evolutionary conservedtranscription factor binding sites. In our test data sets it successfully predicts target transcription factors and their binding sites.

C-to-U editing modifies 30-40 distinct nucleotides within higher-plant chloroplast transcripts. Many C targets are located at the same position in homologous genes from different plants; these either could have emerged independently or could share a common origin. The 5' sequence GCCGUU, required for editing of C214 in tobacco psbE in vitro, is one of the few identified editing cis-elements. We investigated psbE sequences from many plant species to determine in what lineage(s) editing of psbE C214 emerged and whether the cis-element identified in tobacco is conserved in plants with a C214. The GCCGUU sequence is present at a high frequency in plants that carry a C214 in psbE. However, Sciadopitys verticillata (Pinophyta) edits C214 despite the presence of nucleotide differences compared to the conserved cis-element. The C214 site in psbE genes is represented in members of four branches of spermatophytes but not in gnetophytes, resulting in the parsimonious prediction that editing of psbE C214 was present in the ancestor of spermatophytes. Extracts from chloroplasts from a species that has a difference in the motif and lacks the C target are incapable of editing tobacco psbE C214 substrates, implying that the critical trans-acting protein factors were not retained without a C target. Because noncoding sequences are less constrained than coding regions, we analyzed sequences 5' to two C editing targets located within coding regions to search for possible editing-related conserved elements. Putative editing cis-elements were uncovered in the 5' UTRs near editing sites psbL C2 and ndhD C2.

Full Text Available The aromatic compound 2,4-dinitrotoluene (DNT, a common impurity in 2,4,6-trinitrotoluene (TNT production, has been suggested as a tracer for the presence of TNT-based landmines due to its stability and high volatility. We have previously described an Escherichia coli bioreporter capable of detecting the presence of DNT vapors, harboring a fusion of the yqjF gene promoter, to a reporter element. However, the DNT metabolite, which is the direct inducer of yqjF, has not yet been identified, nor has the regulatory mechanism of the induction been clarified. We demonstrate here that the YhaJ protein, a member of the LysR type family, acts as a transcriptional regulator of yqjF activation, as well as of a panel of additional E. coli genes. This group of genes share a common sequence motif in their promoters, which is suggested here as a putative YhaJ-box. In addition, we have linked YhaJ to the regulation of quinol-like compound degradation in the cell, and identified yhaK as playing a role in the degradation of DNT.

Full Text Available Abstract Background Much of the morphological diversity in eukaryotes results from differential regulation of gene expression in which transcription factors (TFs play a central role. The nematode Caenorhabditis elegans is an established model organism for the study of the roles of TFs in controlling the spatiotemporal pattern of gene expression. Using the fully sequenced genomes of three Caenorhabditid nematode species as well as genome information from additional more distantly related organisms (fruit fly, mouse, and human we sought to identify orthologous TFs and characterized their patterns of evolution. Results We identified 988 TF genes in C. elegans, and inferred corresponding sets in C. briggsae and C. remanei, containing 995 and 1093 TF genes, respectively. Analysis of the three gene sets revealed 652 3-way reciprocal 'best hit' orthologs (nematode TF set, approximately half of which are zinc finger (ZF-C2H2 and ZF-C4/NHR types and HOX family members. Examination of the TF genes in C. elegans and C. briggsae identified the presence of significant tandem clustering on chromosome V, the majority of which belong to ZF-C4/NHR family. We also found evidence for lineage-specific duplications and rapid evolution of many of the TF genes in the two species. A search of the TFs conserved among nematodes in Drosophila melanogaster, Mus musculus and Homo sapiens revealed 150 reciprocal orthologs, many of which are associated with important biological processes and human diseases. Finally, a comparison of the sequence, gene interactions and function indicates that nematode TFs conserved across phyla exhibit significantly more interactions and are enriched in genes with annotated mutant phenotypes compared to those that lack orthologs in other species. Conclusion Our study represents the first comprehensive genome-wide analysis of TFs across three nematode species and other organisms. The findings indicate substantial conservation of transcription

The transition between the inactive T-state (apoenzyme) and active R-state (effector bound enzyme) of Trypanosoma cruzi pyruvate kinase (PYK) is accompanied by a symmetrical 8° rigid body rocking motion of the A- and C-domain cores in each of the four subunits, coupled with the formation of additional salt bridges across two of the four subunit interfaces. These salt bridges provide increased tetramer stability correlated with an enhanced specificity constant (k cat/S 0.5). A detailed kinetic and structural comparison between the potential drug target PYKs from the pathogenic protists T. cruzi, T. brucei and Leishmania mexicana shows that their allosteric mechanism is conserved. By contrast, a structural comparison of trypanosomatid PYKs with the evolutionarily divergent PYKs of humans and of bacteria shows that they have adopted different allosteric strategies. The underlying principle in each case is to maximize (k cat/S 0.5) by stabilizing and rigidifying the tetramer in an active R-state conformation. However, bacterial and mammalian PYKs have evolved alternative ways of locking the tetramers together. In contrast to the divergent allosteric mechanisms, the PYK active sites are highly conserved across species. Selective disruption of the varied allosteric mechanisms may therefore provide a useful approach for the design of species-specific inhibitors.

Full Text Available Multisubunit RNA polymerases (msRNAPs exhibit high sequence and structural homology, especially within their active sites, which is generally thought to result in msRNAP functional conservation. However, we show that mutations in the trigger loop (TL in the largest subunit of RNA polymerase I (Pol I yield phenotypes unexpected from studies of Pol II. For example, a well-characterized gain-of-function mutation in Pol II results in loss of function in Pol I (Pol II: rpb1- E1103G; Pol I: rpa190-E1224G. Studies of chimeric Pol II enzymes hosting Pol I or Pol III TLs suggest that consequences of mutations that alter TL dynamics are dictated by the greater enzymatic context and not solely the TL sequence. Although the rpa190-E1224G mutation diminishes polymerase activity, when combined with mutations that perturb Pol I catalysis, it enhances polymerase function, similar to the analogous Pol II mutation. These results suggest that Pol I and Pol II have different rate-limiting steps.

Full Text Available PadR-like transcriptional regulators form a structurally-related family of proteins that control the expression of genes associated with detoxification, virulence and multi-drug resistance in bacteria. Only a few members of this family have been studied by genetic, biochemical and biophysical methods, and their structure/function relationships are still largely undefined. Here, we report the crystal structures of two PadR-like proteins from Bacillus cereus, which we named bcPadR1 and bcPadR2 (products of gene loci BC4206 and BCE3449 in strains ATCC 14579 and ATCC 10987, respectively. BC4206, together with its neighboring gene BC4207, was previously shown to become significantly upregulated in presence of the bacteriocin AS-48. DNA mobility shift assays reveal that bcPadR1 binds to a 250 bp intergenic region containing the putative BC4206-BC4207 promoter sequence, while in-situ expression of bcPadR1 decreases bacteriocin tolerance, together suggesting a role for bcPadR1 as repressor of BC4206-BC4207 transcription. The function of bcPadR2 (48% identical in sequence to bcPadR1 is unknown, but the location of its gene just upstream from genes encoding a putative antibiotic ABC efflux pump, suggests a role in regulating antibiotic resistance. The bcPadR proteins are structurally similar to LmrR, a PadR-like transcription regulator in Lactococcus lactis that controls expression of a multidrug ABC transporter via a mechanism of multidrug binding and induction. Together these proteins define a subfamily of conserved, relatively small PadR proteins characterized by a single C-terminal helix for dimerization. Unlike LmrR, bcPadR1 and bcPadR2 lack a central pore for ligand binding, making it unclear whether the transcriptional regulatory roles of bcPadR1 and bcPadR2 involve direct ligand recognition and induction.

PadR-like transcriptional regulators form a structurally-related family of proteins that control the expression of genes associated with detoxification, virulence and multi-drug resistance in bacteria. Only a few members of this family have been studied by genetic, biochemical and biophysical methods, and their structure/function relationships are still largely undefined. Here, we report the crystal structures of two PadR-like proteins from Bacillus cereus, which we named bcPadR1 and bcPadR2 (products of gene loci BC4206 and BCE3449 in strains ATCC 14579 and ATCC 10987, respectively). BC4206, together with its neighboring gene BC4207, was previously shown to become significantly upregulated in presence of the bacteriocin AS-48. DNA mobility shift assays reveal that bcPadR1 binds to a 250 bp intergenic region containing the putative BC4206-BC4207 promoter sequence, while in-situ expression of bcPadR1 decreases bacteriocin tolerance, together suggesting a role for bcPadR1 as repressor of BC4206-BC4207 transcription. The function of bcPadR2 (48% identical in sequence to bcPadR1) is unknown, but the location of its gene just upstream from genes encoding a putative antibiotic ABC efflux pump, suggests a role in regulating antibiotic resistance. The bcPadR proteins are structurally similar to LmrR, a PadR-like transcription regulator in Lactococcus lactis that controls expression of a multidrug ABC transporter via a mechanism of multidrug binding and induction. Together these proteins define a subfamily of conserved, relatively small PadR proteins characterized by a single C-terminal helix for dimerization. Unlike LmrR, bcPadR1 and bcPadR2 lack a central pore for ligand binding, making it unclear whether the transcriptional regulatory roles of bcPadR1 and bcPadR2 involve direct ligand recognition and induction.

As an equilibrium refinement of the Nash equilibrium, evolutionarily stable strategy (ESS) is a key concept in evolutionary game theory and has attracted growing interest. An ESS can be either a pure strategy or a mixed strategy. Even though the randomness is allowed in mixed strategy, the selection probability of pure strategy in a mixed strategy may fluctuate due to the impact of many factors. The fluctuation can lead to more uncertainty. In this paper, such uncertainty involved in mixed strategy has been further taken into consideration: a belief strategy is proposed in terms of Dempster-Shafer evidence theory. Furthermore, based on the proposed belief strategy, a belief-based ESS has been developed. The belief strategy and belief-based ESS can reduce to the mixed strategy and mixed ESS, which provide more realistic and powerful tools to describe interactions among agents.

Mapping the transcription start points of the eap, emp, and vwb promoters revealed a conserved octanucleotide sequence (COS). Deleting this sequence abolished the expression of eap, emp, and vwb. However, electrophoretic mobility shift assays gave no evidence that this sequence was a binding site for SarA or SaeR, known regulators of eap and emp.

Full Text Available Conidia are primary means of asexual reproduction and dispersal in a variety of pathogenic fungi, and it is widely recognized that they play a critical role in animal and plant disease epidemics. However, genetic mechanisms associated with conidiogenesis are complex and remain largely undefined in numerous pathogenic fungi. We previously showed that Htf1, a homeobox transcription factor, is required for conidiogenesis in the rice pathogen Magnaporthe oryzae. In this study, our aim was to characterize how Htf1 homolog regulates common and also distinctive conidiogenesis in three key Fusarium pathogens: F. graminearm, F. verticillioides, and F. oxysporum. When compared to wild-type progenitors, the gene-deletion mutants in Fusarium species failed to form conventional phialides. Rather, they formed clusters of aberrant phialides that resembled elongated hyphae segments, and it is conceivable that this led to the obstruction of conidiation in phialides. We also observed that mutants, as well as wild-type Fusaria, can initiate alternative macroconidia production directly from hyphae through budding-like mechanism albeit at low frequencies. Microscopic observations led us to conclude that proper basal cell division and subsequent foot cell development of macroconidia were negatively impacted in the mutants. In F. verticillioides and F. oxysporum, mutants exhibited a 2- to 5- microconidia complex at the apex of monophialides resulting in a floral petal-like shape. Also, prototypical microconidia chains were absent in F. verticillioides mutants. F. graminearum and F. verticillioides mutants were complemented by introducing its native HTF1 gene or homologs from other Fusarium species. These results suggest that Fusarium Htf1 is functionally conserved homeobox transcription factor that regulates phialide development and conidiogenesis via distinct signaling pathways yet to be characterized in fungi.

Using an analytical model, it was shown that for a given amount of nitrogen in the canopy of a stand (N-T), there exists an evolutionarily stable leaf area index (ES-LAI), and therefore an evolutionarily stable average leaf nitrogen content (n(anu)(ES); n(anu)(ES) = N-T/ES - LAI), at which no indivi

Mammalian ZFY genes are located on the Y chromosome, and code putative transcription factors with 12-13 zinc fingers preceded by a large acidic (activating) domain. In mice, there are two genes, Zfy1 and Zfy2, which are expressed mainly in the testis. Their transcription increases in germ cells as they enter meiosis, both are silenced by meiotic sex chromosome inactivation (MSCI) during pachytene, and Zfy2 is strongly reactivated later in spermatids. Recently, we have shown that mouse Zfy2, but not Zfy1, is involved in triggering the apoptotic elimination of specific types of sex chromosomally aberrant spermatocytes. In humans, there is a single widely transcribed ZFY gene, and there is no evidence for a specific role in the testis. Here, we characterize ZFY transcription during spermatogenesis in mice and humans. In mice, we define a variety of Zfy transcripts, among which is a Zfy2 transcript that predominates in spermatids, and a Zfy1 transcript, lacking an exon encoding approximately half of the acidic domain, which predominates prior to MSCI. In humans, we have identified a major testis-specific ZFY transcript that encodes a protein with the same short acidic domain. This represents the first evidence that ZFY has a conserved function during human spermatogenesis. We further show that, in contrast to the full acidic domain, the short domain does not activate transcription in yeast, and we hypothesize that this explains the functional difference observed between Zfy1 and Zfy2 during mouse meiosis.

Bathynellacea (Crustacea, Syncarida, Parabathynellidae) are subterranean aquatic crustaceans that typically inhabit freshwater interstitial spaces (e.g., groundwater) and are occasionally found in caves and even hot springs. In this study, we sequenced the whole transcriptome of Allobathynella bangokensis using RNA-seq. De novo sequence assembly produced 74,866 contigs including 28,934 BLAST hits. Overall, the gene sequences were most similar to those of the waterflea Daphnia pulex. In the A. bangokensis transcriptome, no opsin or related sequences were identified, and no contig aligned to the crustacean visual opsins and non-visual opsins (i.e. arthropsins, peropsins, and melaopsins), suggesting potential regressive adaptation to the dark environment. However, A. bangokensis expressed conserved gene family sets, such as heat shock proteins and those related to key innate immunity pathways and antioxidant defense systems, at the transcriptional level, suggesting that this species has evolved adaptations involving molecular mechanisms of homeostasis. The transcriptomic information of A. bangokensis will be useful for investigating molecular adaptations and response mechanisms to subterranean environmental conditions. PMID:28107438

Full Text Available Abstract Background The NAC transcription factor family is involved in the regulation of traits in both monocots and dicots of high agronomic importance. Understanding the precise functions of the NAC genes can be of utmost importance for the improvement of cereal crop plants through plant breeding. For the cereal crop plant barley (Hordeum vulgare L. only a few NAC genes have so far been investigated. Results Through searches in publicly available barley sequence databases we have obtained a list of 48 barley NAC genes (HvNACs with 43 of them representing full-length coding sequences. Phylogenetic comparisons to Brachypodium, rice, and Arabidopsis NAC proteins indicate that the barley NAC family includes members from all of the eight NAC subfamilies, although by comparison to these species a number of HvNACs still remains to be identified. Using qRT-PCR we investigated the expression profiles of 46 HvNACs across eight barley tissues (young flag leaf, senescing flag leaf, young ear, old ear, milk grain, late dough grain, roots, and developing stem and two hormone treatments (abscisic acid and methyl jasmonate. Conclusions Comparisons of expression profiles of selected barley NAC genes with the published functions of closely related NAC genes from other plant species, including both monocots and dicots, suggest conserved functions in the areas of secondary cell wall biosynthesis, leaf senescence, root development, seed development, and hormone regulated stress responses.

A common function of the TFIID and SAGA complexes, which are recruited by transcriptional activators, is to deliver TBP to promoters to stimulate transcription. Neither the relative contributions of the five shared TBP-associated factor (TAF) subunits in TFIID and SAGA nor the requirement for different domains in shared TAFs for transcriptional activation is well understood. In this study, we uncovered the essential requirement for the highly conserved C-terminal region (CRD) of Taf9, a shared TAF, for transcriptional activation in yeast. Transcriptome profiling performed under Gcn4-activating conditions showed that the Taf9 CRD is required for induced expression of ∼9% of the yeast genome. The CRD was not essential for the Taf9-Taf6 interaction, TFIID or SAGA integrity, or Gcn4 interaction with SAGA in cell extracts. Microarray profiling of a SAGA mutant (spt20Δ) yielded a common set of genes induced by Spt20 and the Taf9 CRD. Chromatin immunoprecipitation (ChIP) assays showed that, although the Taf9 CRD mutation did not impair Gcn4 occupancy, the occupancies of TFIID, SAGA, and the preinitiation complex were severely impaired at several promoters. These results suggest a crucial role for the Taf9 CRD in genome-wide transcription and highlight the importance of conserved domains, other than histone fold domains, as a common determinant for TFIID and SAGA functions.

The Nav1.7 voltage-gated sodium channel, encoded by SCN9A, is critical for human pain perception yet the transcriptional and post-transcriptional mechanisms that regulate this gene are still incompletely understood. Here, we describe a novel natural antisense transcript (NAT) for SCN9A that is conse

The endosymbiotic association between legumes and soil bacteria called rhizobia leads to the formation of a new root-derived organ called the nodule in which differentiated bacteria convert atmospheric nitrogen into a form that can be assimilated by the host plant. Successful root infection by rhizobia and nodule organogenesis require the activation of symbiotic genes that are controlled by a set of transcription factors (TFs). We recently identified Medicago truncatula nuclear factor-YA1 (MtNF-YA1) and MtNF-YA2 as two M. truncatula TFs playing a central role during key steps of the Sinorhizobium meliloti-M. truncatula symbiotic interaction. NF-YA TFs interact with NF-YB and NF-YC subunits to regulate target genes containing the CCAAT box consensus sequence. In this study, using a yeast two-hybrid screen approach, we identified the NF-YB and NF-YC subunits able to interact with MtNF-YA1 and MtNF-YA2. In yeast (Saccharomyces cerevisiae) and in planta, we further demonstrated by both coimmunoprecipitation and bimolecular fluorescence complementation that these NF-YA, -B, and -C subunits interact and form a stable NF-Y heterotrimeric complex. Reverse genetic and chromatin immunoprecipitation-PCR approaches revealed the importance of these newly identified NF-YB and NF-YC subunits for rhizobial symbiosis and binding to the promoter of MtERN1 (for Ethylene Responsive factor required for Nodulation), a direct target gene of MtNF-YA1 and MtNF-YA2. Finally, we verified that a similar trimer is formed in planta by the common bean (Phaseolus vulgaris) NF-Y subunits, revealing the existence of evolutionary conserved NF-Y protein complexes to control nodulation in leguminous plants. This sheds light on the process whereby an ancient heterotrimeric TF mainly controlling cell division in animals has acquired specialized functions in plants.

Highlights: ► Chimpanzees, horses and humans have regions of similarity on TLR4 and MD2 promoters. ► Rodents have few regions of similarity on TLR4 promoter when compared to primates. ► Conserved NFkB binding sites were found in the promoters of TLR4 and MD2. ► LPS-induced inhibition of TLR4 transcription is reversed by dexamethasone. ► LPS-induced transcription of MD2 is inhibited by dexamethasone. -- Abstract: Engagement of Toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS) is a master trigger of the deleterious effects of septic shock. Horses and humans are considered the most sensitive species to septic shock, but the mechanisms explaining these phenomena remain elusive. Analysis of tlr4 promoters revealed high similarity among LPS-sensitive species (human, chimpanzee, and horse) and low similarity with LPS-resistant species (mouse and rat). Four conserved nuclear factor kappa B (NFκB) binding sites were found in the tlr4 promoter and two in the md2 promoter sequences that are likely to be targets for dexamethasone regulation. In vitro treatment of equine peripheral blood mononuclear cells (eqPBMC) with LPS decreased transcripts of tlr4 and increased transcription of md2 (myeloid differentiation factor 2) and cd14 (cluster of differentiation 14). Treatment with dexamethasone rescued transcription of tlr4 after LPS inhibition. LPS-induced transcription of md2 was inhibited in the presence of dexamethasone. Dexamethasone alone did not affect transcription of tlr4 and md2.

Full Text Available Abstract Background The transition metal molybdenum is essential for life. Escherichia coli imports this metal into the cell in the form of molybdate ions, which are taken up via an ABC transport system. In E. coli and other Proteobacteria molybdenum metabolism and homeostasis are regulated by the molybdate-responsive transcription factor ModE. Results Orthologues of ModE are widespread amongst diverse prokaryotes, but not ubiquitous. We identified probable ModE-binding sites upstream of genes implicated in molybdenum metabolism in green sulphur bacteria and methanogenic Archaea as well as in Proteobacteria. We also present evidence of horizontal transfer of nitrogen fixation genes between green sulphur bacteria and methanogenic Archaea. Conclusions Whereas most of the archaeal helix-turn-helix-containing transcription factors belong to families that are Archaea-specific, ModE is unusual in that it is found in both Archaea and Bacteria. Moreover, its cognate upstream DNA recognition sequence is also conserved between Archaea and Bacteria, despite the fundamental differences in their core transcription machinery. ModE is the third example of a transcriptional regulator with a binding signal that is conserved in Bacteria and Archaea.

The identification of sequences that control transcription in metazoans is a major goal of genome analysis. In a previous study, we demonstrated that searching for clusters of predicted transcription factor binding sites could discover active regulatory sequences, and identified 37 regions of the Drosophila melanogaster genome with high densities of predicted binding sites for five transcription factors involved in anterior-posterior embryonic patterning. Nine of these clusters overlapped known enhancers. Here, we report the results of in vivo functional analysis of 27 remaining clusters. We generated transgenic flies carrying each cluster attached to a basal promoter and reporter gene, and assayed embryos for reporter gene expression. Six clusters are enhancers of adjacent genes: giant, fushi tarazu, odd-skipped, nubbin, squeeze and pdm2; three drive expression in patterns unrelated to those of neighboring genes; the remaining 18 do not appear to have enhancer activity. We used the Drosophila pseudoobscura genome to compare patterns of evolution in and around the 15 positive and 18 false-positive predictions. Although conservation of primary sequence cannot distinguish true from false positives, conservation of binding-site clustering accurately discriminates functional binding-site clusters from those with no function. We incorporated conservation of binding-site clustering into a new genome-wide enhancer screen, and predict several hundred new regulatory sequences, including 85 adjacent to genes with embryonic patterns. Measuring conservation of sequence features closely linked to function--such as binding-site clustering--makes better use of comparative sequence data than commonly used methods that examine only sequence identity.

The urokinase-type plasminogen activator receptor (uPAR) focuses at the cell surface the activation of pro-uPA and, hence, the formation of plasmin, thus enhancing directional extracellular proteolysis. To characterize the transcriptional regulatory mechanisms that control receptor expression, we...... have cloned an uPAR DNA segment containing upstream regulatory sequences from both the human and murine genomes. We report that a proximal promoter, contained within 180 bp from the major transcription start sites of the human uPAR gene, drives basal transcription. This region lacks TATA and CAAT boxes...

data, we performed an evolutionary and structural characterization of plant F-bZIPs. We observed divergence during seed plant evolution, into two groups and inferred different selective pressures for each. Group 1 contains AtbZIP19 and AtbZIP23 and appears more conserved, whereas Group 2, containing...... be the result of neo-functionalization, the AtbZIP19/23 function in the regulation of the zinc deficiency response may be conserved in land plants (Embryophytes)....

Full Text Available Abstract Background Gene expression analyses have led to a better understanding of growth control of prostate cancer cells. We and others have identified the presence of several zinc finger transcription factors in the neoplastic prostate, suggesting a potential role for these genes in the regulation of the prostate cancer transcriptome. One of the transcription factors (TFs identified in the prostate cancer epithelial cells was the Wilms tumor gene (WT1. To rapidly identify coordinately expressed prostate cancer growth control genes that may be regulated by WT1, we used an in silico approach. Results Evolutionary conservedtranscription factor binding sites (TFBS recognized by WT1, EGR1, SP1, SP2, AP2 and GATA1 were identified in the promoters of 24 differentially expressed prostate cancer genes from eight mammalian species. To test the relationship between sequence conservation and function, chromatin of LNCaP prostate cancer and kidney 293 cells were tested for TF binding using chromatin immunoprecipitation (ChIP. Multiple putative TFBS in gene promoters of placental mammals were found to be shared with those in human gene promoters and some were conserved between genomes that diverged about 170 million years ago (i.e., primates and marsupials, therefore implicating these sites as candidate binding sites. Among those genes coordinately expressed with WT1 was the kallikrein-related peptidase 3 (KLK3 gene commonly known as the prostate specific antigen (PSA gene. This analysis located several potential WT1 TFBS in the PSA gene promoter and led to the rapid identification of a novel putative binding site confirmed in vivo by ChIP. Conversely for two prostate growth control genes, androgen receptor (AR and vascular endothelial growth factor (VEGF, known to be transcriptionally regulated by WT1, regulatory sequence conservation was observed and TF binding in vivo was confirmed by ChIP. Conclusion Overall, this targeted approach rapidly identified

PadR-like transcriptional regulators form a structurally-related family of proteins that control the expression of genes associated with detoxification, virulence and multi-drug resistance in bacteria. Only a few members of this family have been studied by genetic, biochemical and biophysical method

Fungal Wor1-like proteins are conservedtranscriptional regulators that are reported to regulate the virulence of several plant pathogenic fungi by affecting the expression of virulence genes. Here, we report the functional analysis of CfWor1, the homologue of Wor1 in Cladosporium fulvum. Δcfwor1 mutants produce sclerotium-like structures and rough hyphae, which are covered with a black extracellular matrix. These mutants do not sporulate and are no longer virulent on tomato. A CE.CfWor1 transformant that constitutively expresses CfWor1 produces fewer spores with altered morphology and is also reduced in virulence. RNA-seq and RT-qrtPCR analyses suggest that reduced virulence of Δcfwor1 mutants is due to global downregulation of transcription, translation and mitochondrial respiratory chain. The reduced virulence of the CE.CfWor1 transformant is likely due to downregulation of effector genes. Complementation of a non-virulent Δfosge1 (Wor1-homologue) mutant of Fusarium oxysporum f. sp. lycopersici with CfWor1 restored expression of the SIX effector genes in this fungus, but not its virulence. Chimeric proteins of CfWor1/FoSge1 also only partially restored defects of the Δfosge1 mutant, suggesting that these transcriptional regulators have functionally diverged. Altogether, our results suggest that CfWor1 primarily regulates development of C. fulvum, which indirectly affects the expression of a subset of virulence genes.

ETS proteins are a group of evolutionarily related, DNA-binding transcriptional factors. These proteins direct gene expression in diverse normal and disease states by binding to specific promoters and enhancers and facilitating assembly of other components of the transcriptional machinery. The highly conserved DNA-binding ETS domain defines the family and is responsible for specific recognition of a common sequence motif, 5′-GGA(A/T)-3′. Attaining specificity for biological regulation in such a family is thus a conundrum. We present the current knowledge of routes to functional diversity and DNA binding specificity, including divergent properties of the conserved ETS and PNT domains, the involvement of flanking structured and unstructured regions appended to these dynamic domains, posttranslational modifications, and protein partnerships with other DNA-binding proteins and coregulators. The review emphasizes recent advances from biochemical and biophysical approaches, as well as insights from genomic studies that detect ETS-factor occupancy in living cells. PMID:21548782

Transcriptional initiation of eukaryotic genes depends on the cooperative interaction of various transcription factors. Using the yeast two-hybrid assay, we have identified the murine Rcd-1 protein as a cofactor of the c-myb proto-oncogene product. Rcd-1 is evolutionarilyconserved among many species, and moreover the yeast homologue CAF40 is part of the carbon catabolite repressor protein transcriptional mediator thought to be involved in the negative regulation of genes transcribed by RNA polymerase II. Rcd-1 is located mainly in the nucleus, and it interacts with c-Myb both in vitro and in vivo. The activation of the myeloid c-myb-specific mim-1 promoter is repressed by Rcd-1. Interestingly, rcd-1 is an erythropoietin regulated gene, which also represses the action of the AP-1 transcription factor on its target genes.

Regulatory factor binding to genomic DNA protects the underlying sequence from cleavage by DNase I, leaving nucleotide-resolution footprints. Using genomic DNase I footprinting across 41 diverse cell and tissue types, we detected 45 million transcription factor occupancy events within regulatory regions, representing differential binding to 8.4 million distinct short sequence elements. Here we show that this small genomic sequence compartment, roughly twice the size of the exome, encodes an expansive repertoire of conserved recognition sequences for DNA-binding proteins that nearly doubles the size of the human cis-regulatory lexicon. We find that genetic variants affecting allelic chromatin states are concentrated in footprints, and that these elements are preferentially sheltered from DNA methylation. High-resolution DNase I cleavage patterns mirror nucleotide-level evolutionary conservation and track the crystallographic topography of protein-DNA interfaces, indicating that transcription factor structure has been evolutionarily imprinted on the human genome sequence. We identify a stereotyped 50-base-pair footprint that precisely defines the site of transcript origination within thousands of human promoters. Finally, we describe a large collection of novel regulatory factor recognition motifs that are highly conserved in both sequence and function, and exhibit cell-selective occupancy patterns that closely parallel major regulators of development, differentiation and pluripotency.

LIM kinase 1 (LIMK1),a cytosolic serine/threonine kinase,regulates actin filament dynamics and reorganization and is involved in neuronal development and brain function.Abnormal expression of LIMK1 is associated with several neurological disorders.In this study,we performed a conservation analysis using Vector NTI (8.0) software.The dualluciferase reporter assay and real-time quantitative RT-PCR were used to assess the protein and mRNA levels of the reporter gene,respectively.We found that a region ranging from nt +884 to +966 in the human LIMK1 3' untranslated region (UTR) was highly conserved in the mouse Limk1 3' UTR and formed a structure containing several loops and stems.Luciferase assay showed that the relative luciferase activity of the mutated construct with the conserved region deleted,pGL4-hLIMK1-3U-M,in SH-SY5Y and HEK-293 cells was only ～60％ of that of the wild-type construct pGL4-hLIMK1-3U,indicating that the conserved region is critical for the reporter gene expression.Real-time quantitative RT-PCR analysis demonstrated that the relative Luc2 mRNA levels in SH-SY5Y and HEK293 cells transfected with pGL4-hLIMK1-3U-M decreased to ～50％ of that in cells transfected with pGL4-hLIMK1-3U,suggesting an important role of the conserved region in maintaining Luc2 mRNA stability.Our study suggests that the conserved region in the LIMK1 3' UTR is involved in regulating LIMK1 expression at the post-transcriptional level,which may help reveal the mechanism underlying the regulation of LIMK1 expression in the central nervous system and explore the relationship between the 3'-UTR mutant and neurological disorders.

Full Text Available Activating transcription factor 1 (ATF1 and the closely related proteins CREB (cyclic AMP resonse element binding protein and CREM (cyclic AMP response element modulator constitute a subfamily of bZIP transcription factors that play critical roles in the regulation of cellular growth, metabolism, and survival. Previous studies demonstrated that CREB is phosphorylated on a cluster of conserved Ser residues, including Ser-111 and Ser-121, in response to DNA damage through the coordinated actions of the ataxia-telangiectasia-mutated (ATM protein kinase and casein kinases 1 and 2 (CK1/2. Here, we show that DNA damage-induced phosphorylation by ATM is a general feature of CREB and ATF1. ATF1 harbors a conserved ATM/CK cluster that is constitutively and stoichiometrically phosphorylated by CK1 and CK2 in asynchronously growing cells. Exposure to DNA damage further induced ATF1 phosphorylation on Ser-51 by ATM in a manner that required prior phosphorylation of the upstream CK residues. Hyperphosphorylated ATF1 showed a 4-fold reduced affinity for CREB-binding protein. We further show that PP2A, in conjunction with its targeting subunit B56gamma, antagonized ATM and CK1/2-dependent phosphorylation of CREB and ATF1 in cellulo. Finally, we show that CK sites in CREB are phosphorylated during cellular growth and that phosphorylation of these residues reduces the threshold of DNA damage required for ATM-dependent phosphorylation of the inhibitory Ser-121 residue. These studies define overlapping and distinct modes of CREB and ATF1 regulation by phosphorylation that may ensure concerted changes in gene expression mediated by these factors.

Full Text Available BACKGROUND: The chicken is an important agricultural and avian-model species. A survey of gene expression in a range of different tissues will provide a benchmark for understanding expression levels under normal physiological conditions in birds. With expression data for birds being very scant, this benchmark is of particular interest for comparative expression analysis among various terrestrial vertebrates. METHODOLOGY/PRINCIPAL FINDINGS: We carried out a gene expression survey in eight major chicken tissues using whole genome microarrays. A global picture of gene expression is presented for the eight tissues, and tissue specific as well as common gene expression were identified. A Gene Ontology (GO term enrichment analysis showed that tissue-specific genes are enriched with GO terms reflecting the physiological functions of the specific tissue, and housekeeping genes are enriched with GO terms related to essential biological functions. Comparisons of structural genomic features between tissue-specific genes and housekeeping genes show that housekeeping genes are more compact. Specifically, coding sequence and particularly introns are shorter than genes that display more variation in expression between tissues, and in addition intergenic space was also shorter. Meanwhile, housekeeping genes are more likely to co-localize with other abundantly or highly expressed genes on the same chromosomal regions. Furthermore, comparisons of gene expression in a panel of five common tissues between birds, mammals and amphibians showed that the expression patterns across tissues are highly similar for orthologous genes compared to random gene pairs within each pair-wise comparison, indicating a high degree of functional conservation in gene expression among terrestrial vertebrates. CONCLUSIONS: The housekeeping genes identified in this study have shorter gene length, shorter coding sequence length, shorter introns, and shorter intergenic regions, there seems

Delayed transcriptional activation of the zygotic genome is a nearly universal phenomenon in metazoans. Immediately following fertilization, development is controlled by maternally deposited products, and it is not until later stages that widespread activation of the zygotic genome occurs. Although the mechanisms driving this genome activation are currently unknown, the transcriptional activator Zelda (ZLD) has been shown to be instrumental in driving this process in Drosophila melanogaster. Here we define functional domains of ZLD required for both DNA binding and transcriptional activation. We show that the C-terminal cluster of four zinc fingers mediates binding to TAGteam DNA elements in the promoters of early expressed genes. All four zinc fingers are required for this activity, and splice isoforms lacking three of the four zinc fingers fail to activate transcription. These truncated splice isoforms dominantly suppress activation by the full-length, embryonically expressed isoform. We map the transcriptional activation domain of ZLD to a central region characterized by low complexity. Despite relatively little sequence conservation within this domain, ZLD orthologs from Drosophila virilis, Anopheles gambiae, and Nasonia vitripennis activate transcription in D. melanogaster cells. Transcriptional activation by these ZLD orthologs suggests that ZLD functions through conserved interactions with a protein cofactor(s). We have identified distinct DNA-binding and activation domains within the critical transcription factor ZLD that controls the initial activation of the zygotic genome.

Sequence analysis of the five genes (gutRMCBA) downstream from the previously described sorbitol-6-phosphate dehydrogenase-encoding Lactobacillus casei gutF gene revealed that they constitute a sorbitol (glucitol) utilization operon. The gutRM genes encode putative regulators, while the gutCBA genes encode the EIIC, EIIBC, and EIIA proteins of a phosphoenolpyruvate-dependent sorbitol phosphotransferase system (PTSGut). The gut operon is transcribed as a polycistronic gutFRMCBA messenger, the expression of which is induced by sorbitol and repressed by glucose. gutR encodes a transcriptional regulator with two PTS-regulated domains, a galactitol-specific EIIB-like domain (EIIBGat domain) and a mannitol/fructose-specific EIIA-like domain (EIIAMtl domain). Its inactivation abolished gut operon transcription and sorbitol uptake, indicating that it acts as a transcriptional activator. In contrast, cells carrying a gutB mutation expressed the gut operon constitutively, but they failed to transport sorbitol, indicating that EIIBCGut negatively regulates GutR. A footprint analysis showed that GutR binds to a 35-bp sequence upstream from the gut promoter. A sequence comparison with the presumed promoter region of gut operons from various firmicutes revealed a GutR consensus motif that includes an inverted repeat. The regulation mechanism of the L. casei gut operon is therefore likely to be operative in other firmicutes. Finally, gutM codes for a conserved protein of unknown function present in all sequenced gut operons. A gutM mutant, the first constructed in a firmicute, showed drastically reduced gut operon expression and sorbitol uptake, indicating a regulatory role also for GutM. PMID:18676710

Full Text Available Proper formation of the mammalian heart requires precise spatiotemporal transcriptional regulation of gene programs in cardiomyocytes. Sophisticated regulatory networks have evolved to not only integrate the activities of distinct transcription factors to control tissue-specific gene programs but also, in many instances, to incorporate multiple members within these transcription factor families to ensure accuracy and specificity in the system. Unsurprisingly, perturbations in this elaborate transcriptional circuitry can lead to severe cardiac abnormalities. Myocyte enhancer factor–2 (MEF2 transcription factor belongs to the evolutionarilyconserved cardiac gene regulatory network. Given its central role in muscle gene regulation and its evolutionary conservation, MEF2 is considered one of only a few core cardiac transcription factors. In addition to its firmly established role as a differentiation factor, MEF2 regulates wide variety of, sometimes antagonistic, cellular processes such as cell survival and death. Vertebrate genomes encode multiple MEF2 family members thereby expanding the transcriptional potential of this core transcription factor in the heart. This review highlights the requirement of the MEF2 family and their orthologs in cardiac development in diverse animal model systems. Furthermore, we describe the recently characterized role of MEF2 in direct reprogramming and genome-wide cardiomyocyte gene regulation. A thorough understanding of the regulatory functions of the MEF2 family in cardiac development and cardiogenomics is required in order to develop effective therapeutic strategies to repair the diseased heart.

The coordinated and dynamic modulation or interaction of genes or proteins acts as an important mechanism used by a cell in functional regulation. Recent studies have shown that many transcriptional networks exhibit a scale-free topology and hierarchical modular architecture. It has also been shown that transcriptional networks or pathways are dynamic and behave only in certain ways and controlled manners in response to disease development, changing cellular conditions, and different environmental factors. Moreover, evolutionarilyconserved and divergent transcriptional modules underline fundamental and species-specific molecular mechanisms controlling disease development or cellular phenotypes. Various computational algorithms have been developed to explore transcriptional networks and modules from gene expression data. In silico studies have also been made to mimic the dynamic behavior of regulatory networks, analyzing how disease or cellular phenotypes arise from the connectivity or networks of genes and their products. Here, we review the recent development in computational biology research on deciphering modular and dynamic behaviors of transcriptional networks, highlighting important findings. We also demonstrate how these computational algorithms can be applied in systems biology studies as on disease, stem cells, and drug discovery.

Full Text Available Transcripts of centromeric satellite DNAs are known to play a role in heterochromatin formation as well as in establishment of the kinetochore. However, little is known about basic mechanisms of satellite DNA expression within constitutive heterochromatin and its regulation. Here we present comprehensive analysis of transcription of abundant centromeric satellite DNA, PRAT from beetle Palorus ratzeburgii (Coleoptera. This satellite is characterized by preservation and extreme sequence conservation among evolutionarily distant insect species. PRAT is expressed in all three developmental stages: larvae, pupae and adults at similar level. Transcripts are abundant comprising 0.033% of total RNA and are heterogeneous in size ranging from 0.5 kb up to more than 5 kb. Transcription proceeds from both strands but with 10 fold different expression intensity and transcripts are not processed into siRNAs. Most of the transcripts (80% are not polyadenylated and remain in the nucleus while a small portion is exported to the cytoplasm. Multiple, irregularly distributed transcription initiation sites as well as termination sites have been mapped within the PRAT sequence using primer extension and RLM-RACE. The presence of cap structure as well as poly(A tails in a portion of the transcripts indicate RNA polymerase II-dependent transcription and a putative polymerase II promoter site overlaps the most conserved part of the PRAT sequence. The treatment of larvae with alpha-amanitin decreases the level of PRAT transcripts at concentrations that selectively inhibit pol II activity. In conclusion, stable, RNA polymerase II dependant transcripts of abundant centromeric satellite DNA, not regulated by RNAi, have been identified and characterized. This study offers a basic understanding of expression of highly abundant heterochromatic DNA which in beetle species constitutes up to 50% of the genome.

Piwi-interacting (pi-) RNAs guide germline-expressed Piwi proteins in order to suppress the activity of transposable elements (TEs). But notably, the majority of pachytene piRNAs in mammalian testes is not related to TEs. This raises the question of whether the Piwi/piRNA pathway exerts functions beyond TE silencing. Although gene-derived piRNAs were described many times, a possible gene-regulatory function was doubted due to the absence of antisense piRNAs. Here we sequenced and analyzed piRNAs expressed in the adult testis of the pig, as this taxon possesses the full set of mammalian Piwi paralogs while their spermatozoa are marked by an extreme fitness due to selective breeding. We provide an exhaustive characterization of porcine piRNAs and genomic piRNA clusters. Moreover, we reveal that both sense and antisense piRNAs derive from protein-coding genes, while exhibiting features that clearly show that they originate from the Piwi/piRNA-mediated post-transcriptional silencing pathway, commonly referred to as ping-pong cycle. We further show that the majority of identified piRNA clusters in the porcine genome spans exonic sequences of protein-coding genes or pseudogenes, which reveals a mechanism by which primary antisense piRNAs directed against mRNA can be generated. Our data provide evidence that spliced mRNAs, derived from such loci, are not only targeted by piRNAs but are also subject to ping-pong cycle processing. Finally, we demonstrate that homologous genes are targeted and processed by piRNAs in pig, mouse and human. Altogether, this strongly suggests a conserved role for the mammalian Piwi/piRNA pathway in post-transcriptional regulation of protein-coding genes, which did not receive much attention so far.

Sonic Hedgehog (Shh) signaling helps pattern the vertebrate neural tube, in part by regulating the dorsal/ventral expression of a number of homeodomain containing transcription factors. These Hh responsive genes have been divided into two classes, with Class II genes being activated by Hh signaling and Class I genes being repressed by Hh signaling. While the transcriptional response to varying Hh levels is well defined in chick and mouse, it is only partially described in zebrafish, despite the fact that zebrafish has emerged as a powerful genetic system for the study of neural patterning. To better characterize the Hh response in the zebrafish neural tube, we cloned the zebrafish Class II Hh target genes nkx2.9 and nkx6.2. We then analyzed the expression of a number of Class I and Class II Hh responsive genes in wild type, Hh mutant, and Hh over-expressing zebrafish embryos. We show that expression of Class I and Class II genes is highly conserved in the vertebrate neural tube. Further, ventral-most Class II gene expression was completely lost in all Hh pathway mutants analyzed, indicating high levels of Hh signaling are blocked in all of these mutants. In contrast, more dorsally expressed genes were variably affected in different Hh pathway mutants, indicating mid-levels of Hh signaling are differentially affected. This comprehensive expression study provides an important tool for the characterization of Hh signaling in zebrafish and provides a sensitive assay for determining the degree to which newly identified zebrafish mutants affect Hh signaling.

Mammalian lipins (lipin-1, lipin-2, and lipin-3) are Mg2+-dependent phosphatidate phosphatase (PAP) enzymes, which catalyze a key reaction in glycerolipid biosynthesis. Lipin-1 also functions as a transcriptional coactivator in conjunction with members of the peroxisome proliferator-activated receptor family. An S734L mutation in LPIN2 causes Majeed syndrome, a human inflammatory disorder characterized by recurrent osteomyelitis, fever, dyserythropoietic anemia, and cutaneous inflammation. Here we demonstrate that mutation of the equivalent serine in mouse lipin-1 and lipin-2 to leucine or aspartate abolishes PAP activity but does not impair lipin association with microsomal membranes, the major site of glycerolipid synthesis. We also determined that lipin-2 has transcriptional coactivator activity for peroxisome proliferator-activated receptor-response elements similar to lipin-1 and that this activity is not affected by mutating the conserved serine. Therefore, our results indicate that the symptoms of the Majeed syndrome result from a loss of lipin-2 PAP activity. To characterize sites of lipin-2 action, we detected lipin-2 expression by in situ hybridization on whole mouse sections and by quantitative PCR of tissues relevant to Majeed syndrome. Lipin-2 was most prominently expressed in liver, where levels were much higher than lipin-1, and also in kidney, lung, gastrointestinal tract, and specific regions of the brain. Lipin-2 was also expressed in circulating red blood cells and sites of lymphopoiesis (bone marrow, thymus, and spleen). These results raise the possibility that the loss of lipin-2 PAP activity in erythrocytes and lymphocytes may contribute to the anemia and inflammation phenotypes observed in Majeed syndrome patients.

Full Text Available Angiotensin converting enzyme (ACE is essential for control of blood pressure. The human ACE gene contains an intronic Alu indel (I/D polymorphism that has been associated with variation in serum enzyme levels, although the functional mechanism has not been identified. The polymorphism has also been associated with cardiovascular disease, type II diabetes, renal disease and elite athleticism. We have characterized the ACE gene in horses of breeds selected for differing physical abilities. The equine gene has a similar structure to that of all known mammalian ACE genes. Nine common single nucleotide polymorphisms (SNPs discovered in pooled DNA were found to be inherited in nine haplotypes. Three of these SNPs were located in intron 16, homologous to that containing the Alu polymorphism in the human. A highly conserved 18 bp sequence, also within that intron, was identified as being a potential binding site for the transcription factors Oct-1, HFH-1 and HNF-3β, and lies within a larger area of higher than normal homology. This putative regulatory element may contribute to regulation of the documented inter-individual variation in human circulating enzyme levels, for which a functional mechanism is yet to be defined. Two equine SNPs occurred within the conserved area in intron 16, although neither of them disrupted the putative binding site. We propose a possible regulatory mechanism of the ACE gene in mammalian species which was previously unknown. This advance will allow further analysis leading to a better understanding of the mechanisms underpinning the associations seen between the human Alu polymorphism and enzyme levels, cardiovascular disease states and elite athleticism.

Full Text Available So far, the computational identification of transcription factor binding sites is hampered by the complexity of vertebrate genomes. Here we present an in silico procedure to predict target sites of a transcription factor in complex genomes using its binding site. In a first step sequence, comparison of closely related genomes identifies the binding sites in conserved cis-regulatory regions (phylogenetic footprinting. Subsequently, more remote genomes are introduced into the comparison to identify highly conserved and therefore putatively functional binding sites (phylogenetic filtering. When applied to the binding site of atonal homolog 5 (Ath5 or ATOH7, this procedure efficiently filters evolutionarilyconserved binding sites out of more than 300,000 instances in a vertebrate genome. We validate a selection of the linked target genes by showing coexpression with and transcriptional regulation by Ath5. Finally, chromatin immunoprecipitation demonstrates the occupancy of the target gene promoters by Ath5. Thus, our procedure, applied to whole genomes, is a fast and predictive tool to in silico filter the target genes of a given transcription factor with defined binding site.

The Hansenula polymorpha MOX gene encodes a peroxisomal enzyme that catalyzes the first step of the highly specialized methanol metabolism. MOX is strongly transcribed in cells growing in methanol and completely repressed in glucose. We show here that the MOX promoter confers a glucose-repressible expression upon a lacZ reporter gene in Saccharomyces cerevisiae, an unrelated yeast species that lacks the methanol metabolism. Repression was mediated by a 200-bp region of the MOX promoter, termed MOX-B, and was counteracted by Adr1p, a transcription factor involved in the derepression of S. cerevisiae genes encoding peroxisomal proteins, the class to which MOX belongs. Binding of Adr1p to MOX-B was demonstrated by gel retardation and DNaseI-footprinting, and Adr1p was shown to interact with a DNA region containing only a half of the putative Adr1p consensus binding site. Our findings suggest that Adr1p is a conserved regulator for genes encoding peroxisomal proteins at least in other yeast species, and that its interaction with the DNA is dependent on the promoter context.

The genes encoding the closely related zinc finger transcription factors Buttonhead (Btd) and D-Sp1 are expressed in the developing limb primordia of Drosophila melanogaster and are required for normal growth of the legs. The D-Sp1 homolog of the red flour beetle Tribolium castaneum, Sp8 (appropriately termed Sp8/9), is also required for the proper growth of the leg segments. Here we report on the isolation and functional study of the Sp8/9 gene from the milkweed bug Oncopeltus fasciatus. We show that Sp8/9 is expressed in the developing appendages throughout development and that the downregulation of Sp8/9 via RNAi leads to antennae, rostrum, and legs with shortened and fused segments. This supports a conserved role of Sp8/9 in allometric leg segment growth. However, all leg segments including the claws are present and the expression of the leg genes Distal-less, dachshund, and homothorax are proportionally normal, thus providing no evidence for a role of Sp8/9 in appendage specification.

Full Text Available Abstract Background Transcription factors activate their target genes by binding to specific response elements. Many transcription factor families evolved from a common ancestor by gene duplication and subsequent divergent evolution. Members of the p53 family, which play key roles in cell-cycle control and development, share conserved DNA binding and oligomerisation domains but exhibit distinct functions. In this study, the molecular basis of the functional divergence of related transcription factors was investigated. Results We characterised the DNA-binding specificity and oligomerisation properties of human p53, p63 and p73, as well as p53 from other organisms using novel biophysical approaches. All p53 family members bound DNA cooperatively as tetramers with high affinity. Despite structural differences in the oligomerisation domain, the dissociation constants of the tetramers was in the low nanomolar range for all family members, indicating that the strength of tetramerisation was evolutionarilyconserved. However, small differences in the oligomerisation properties were observed, which may play a regulatory role. Intriguingly, the DNA-binding specificity of p53 family members was highly conserved even for evolutionarily distant species. Additionally, DNA recognition was only weakly affected by CpG methylation. Prediction of p53/p63/p73 binding sites in the genome showed almost complete overlap between the different homologs. Conclusion Diversity of biological function of p53 family members is not reflected in differences in sequence-specific DNA binding. Hence, additional specificity factors must exist, which allowed the acquisition of novel functions during evolution while preserving original roles.

Nicotinamide adenine dinucleotide phosphate (NADP) is a critical cofactor during metabolism, calcium signaling, and oxidative defense, yet how animals regulate their NADP pools in vivo and how NADP-synthesizing enzymes are regulated have long remained unknown. Here we show that expression of Nadk, an NAD(+) kinase-encoding gene, governs NADP biosynthesis in vivo and is essential for development in Xenopus frog embryos. Unexpectedly, we found that embryonic Nadk expression is dynamic, showing cell type-specific up-regulation during both frog and sea urchin embryogenesis. We analyzed the NAD kinases (NADKs) of a variety of deuterostome animals, finding two conserved internal domains forming a catalytic core but a highly divergent N terminus. One type of N terminus (found in basal species such as the sea urchin) mediates direct catalytic activation of NADK by Ca(2+)/calmodulin (CaM), whereas the other (typical for vertebrates) is phosphorylated by a CaM kinase-dependent mechanism. This work indicates that animal NADKs govern NADP biosynthesis in vivo and are regulated by evolutionarily divergent and conserved CaM-dependent mechanisms.

The collagenase matrix metalloproteinase-13 (MMP-13) plays an important role in the destruction of cartilage in arthritic joints. MMP-13 expression is strongly up-regulated in arthritis, largely because of stimulation by inflammatory cytokines such as IL-1β. Treatment of chondrocytes with IL-1β induces transcription of MMP-13 in vitro. IL-1β signaling converges upon the activator protein-1 transcription factors, which have been shown to be required for IL-1β-induced MMP-13 gene expression. Using chromatin immunoprecipitation (ChIP), we detected activator protein-1 binding within an evolutionarilyconserved DNA sequence ∼20 kb 5′ relative to the MMP-13 transcription start site (TSS). Also using ChIP, we detected histone modifications and binding of RNA polymerase II within this conserved region, all of which are consistent with transcriptional activation. Chromosome conformation capture indicates that chromosome looping brings this region in close proximity with the MMP-13 TSS. Finally, a luciferase reporter construct driven by a component of the conserved region demonstrated an expression pattern similar to that of endogenous MMP-13. These data suggest that a conserved region at 20 kb upstream from the MMP-13 TSS includes a distal transcriptional response element of MMP-13, which contributes to MMP-13 gene expression. PMID:22102411

The maintenance of cellular proteins in a biologically active and structurally stable state is a vital endeavor involving multiple cellular pathways. One such pathway is the ubiquitin-proteasome system that represents a major route for protein degradation, and reductions in this pathway usually have adverse effects on the health of cells and tissues. Here, we demonstrate that loss-of-function mutants of the Caenorhabditis elegans proteasome subunit, RPN-10, exhibit moderate proteasome dysfunction and unexpectedly develop both increased longevity and enhanced resistance to multiple threats to the proteome, including heat, oxidative stress, and the presence of aggregation prone proteins. The rpn-10 mutant animals survive through the activation of compensatory mechanisms regulated by the conserved SKN-1/Nrf2 and ELT-2/GATA transcription factors that mediate the increased expression of genes encoding proteasome subunits as well as those mediating oxidative- and heat-stress responses. Additionally, we find that the rpn-10 mutant also shows enhanced activity of the autophagy-lysosome pathway as evidenced by increased expression of the multiple autophagy genes including atg-16.2, lgg-1, and bec-1, and also by an increase in GFP::LGG-1 puncta. Consistent with a critical role for this pathway, the enhanced resistance of the rpn-10 mutant to aggregation prone proteins depends on autophagy genes atg-13, atg-16.2, and prmt-1. Furthermore, the rpn-10 mutant is particularly sensitive to the inhibition of lysosome activity via either RNAi or chemical means. We also find that the rpn-10 mutant shows a reduction in the numbers of intestinal lysosomes, and that the elt-2 gene also plays a novel and vital role in controlling the production of functional lysosomes by the intestine. Overall, these experiments suggest that moderate proteasome dysfunction could be leveraged to improve protein homeostasis and organismal health and longevity, and that the rpn-10 mutant provides a unique

Full Text Available The maintenance of cellular proteins in a biologically active and structurally stable state is a vital endeavor involving multiple cellular pathways. One such pathway is the ubiquitin-proteasome system that represents a major route for protein degradation, and reductions in this pathway usually have adverse effects on the health of cells and tissues. Here, we demonstrate that loss-of-function mutants of the Caenorhabditis elegans proteasome subunit, RPN-10, exhibit moderate proteasome dysfunction and unexpectedly develop both increased longevity and enhanced resistance to multiple threats to the proteome, including heat, oxidative stress, and the presence of aggregation prone proteins. The rpn-10 mutant animals survive through the activation of compensatory mechanisms regulated by the conserved SKN-1/Nrf2 and ELT-2/GATA transcription factors that mediate the increased expression of genes encoding proteasome subunits as well as those mediating oxidative- and heat-stress responses. Additionally, we find that the rpn-10 mutant also shows enhanced activity of the autophagy-lysosome pathway as evidenced by increased expression of the multiple autophagy genes including atg-16.2, lgg-1, and bec-1, and also by an increase in GFP::LGG-1 puncta. Consistent with a critical role for this pathway, the enhanced resistance of the rpn-10 mutant to aggregation prone proteins depends on autophagy genes atg-13, atg-16.2, and prmt-1. Furthermore, the rpn-10 mutant is particularly sensitive to the inhibition of lysosome activity via either RNAi or chemical means. We also find that the rpn-10 mutant shows a reduction in the numbers of intestinal lysosomes, and that the elt-2 gene also plays a novel and vital role in controlling the production of functional lysosomes by the intestine. Overall, these experiments suggest that moderate proteasome dysfunction could be leveraged to improve protein homeostasis and organismal health and longevity, and that the rpn-10 mutant

Full Text Available BACKGROUND: microRNAs (miRNAs have been reported as key regulators at post-transcriptional level in eukaryotic cells. In insects, most of the studies have focused in holometabolans while only recently two hemimetabolans (Locusta migratoria and Acyrthosiphon pisum have had their miRNAs identified. Therefore, the study of the miRNAs of the evolutionarily basal hemimetabolan Blattella germanica may provide valuable insights on the structural and functional evolution of miRNAs. METHODOLOGY/PRINCIPAL FINDINGS: Small RNA libraries of the cockroach B. germanica were built from the whole body of the last instar nymph, and the adult ovaries. The high throughput Solexa sequencing resulted in approximately 11 and 8 million reads for the whole-body and ovaries, respectively. Bioinformatic analyses identified 38 known miRNAs as well as 11 known miRNA*s. We also found 70 miRNA candidates conserved in other insects and 170 candidates specific to B. germanica. The positive correlation between Solexa data and real-time quantitative PCR showed that number of reads can be used as a quantitative approach. Five novel miRNA precursors were identified and validated by PCR and sequencing. Known miRNAs and novel candidates were also validated by decreasing levels of their expression in dicer-1 RNAi knockdown individuals. The comparison of the two libraries indicates that whole-body nymph contain more known miRNAs than ovaries, whereas the adult ovaries are enriched with novel miRNA candidates. CONCLUSIONS/SIGNIFICANCE: Our study has identified many known miRNAs and novel miRNA candidates in the basal hemimetabolan insect B. germanica, and most of the specific sequences were found in ovaries. Deep sequencing data reflect miRNA abundance and dicer-1 RNAi assay is shown to be a reliable method for validation of novel miRNAs.

The way in which the network of intramolecular interactions determines the cooperative folding and conformational dynamics of a protein remains poorly understood. High-pressure NMR spectroscopy is uniquely suited to examine this problem because it combines the site-specific resolution of the NMR experiments with the local character of pressure perturbations. Here we report on the temperature dependence of the site-specific volumetric properties of various forms of staphylococcal nuclease (SNase), including three variants with engineered internal cavities, as measured with high-pressure NMR spectroscopy. The strong temperature dependence of pressure-induced unfolding arises from poorly understood differences in thermal expansion between the folded and unfolded states. A significant inverse correlation was observed between the global thermal expansion of the folded proteins and the number of strong intramolecular hydrogen bonds, as determined by the temperature coefficient of the backbone amide chemical shifts. Comparison of the identity of these strong H-bonds with the co-evolution of pairs of residues in the SNase protein family suggests that the architecture of the interactions detected in the NMR experiments could be linked to a functional aspect of the protein. Moreover, the temperature dependence of the residue-specific volume changes of unfolding yielded residue-specific differences in expansivity and revealed how mutations impact intramolecular interaction patterns. These results show that intramolecular interactions in the folded states of proteins impose constraints against thermal expansion and that, hence, knowledge of site-specific thermal expansivity offers insight into the patterns of strong intramolecular interactions and other local determinants of protein stability, cooperativity, and potentially also of function.

Full Text Available Abstract Background Hematopoiesis is a complex developmental process controlled by a large number of factors that regulate stem cell renewal, lineage commitment and differentiation. Secreted proteins, including the hematopoietic growth factors, play critical roles in these processes and have important biological and clinical significance. We have employed representational difference analysis to identify genes that are differentially expressed during experimentally induced myeloid differentiation in the murine EML hematopoietic stem cell line. Results One identified clone encoded a previously unidentified protein of 541 amino acids that contains an amino terminal signal sequence but no other characterized domains. This protein is a member of family of related proteins that has been named family with sequence similarity 20 (FAM20 with three members (FAM20A, FAM20B and FAM20C in mammals. Evolutionary comparisons revealed the existence of a single FAM20 gene in the simple vertebrate Ciona intestinalis and the invertebrate worm Caenorhabditis elegans and two genes in two insect species, Drosophila melanogaster and Anopheles gambiae. Six FAM20 family members were identified in the genome of the pufferfish, Fugu rubripes and five members in the zebrafish, Danio rerio. The mouse Fam20a protein was ectopically expressed in a mammalian cell line and found to be a bona fide secreted protein and efficient secretion was dependent on the integrity of the signal sequence. Expression analysis revealed that the Fam20a gene was indeed differentially expressed during hematopoietic differentiation and that the other two family members (Fam20b and Fam20c were also expressed during hematcpoiesis but that their mRNA levels did not vary significantly. Likewise FAM20A was expressed in more limited set of human tissues than the other two family members. Conclusions The FAM20 family represents a new family of secreted proteins with potential functions in regulating differentiation and function of hematopoietic and other tissues. The Fam20a mRNA was only expressed during early stages of hematopoietic development and may play a role in lineage commitment or proliferation. The expansion in gene number in different species suggests that the family has evolved as a result of several gene duplication events that have occurred in both vertebrates and invertebrates.

Rationale: Lymphatic vessel formation and function constitutes a physiologically and pathophysiologically important process, but its genetic control is not well understood. Objective: Here, we identify the secreted Polydom/Svep1 protein as essential for the formation of the lymphatic vasculature. We

Little is known about the potential threats of silver nanoparticles (AgNPs) to ecosystem health, with no detailed report existing on the stress and immune responses of soil invertebrates. Here we use earthworm primary cells, cross-referencing to human cell cultures with a particular emphasis on t...

The role of serotonin (5-HT) in psychopathology has been investigated for decades. Among others, symptoms of depression, panic, aggression and suicidality have been associated with serotonergic dysfunction. Here we summarize the evidence that low brain 5-HT signals a metabolic imbalance that is evol

Sir2 is a NAD+-dependent protein deacetylase that extends lifespan in yeast and worms. This study examines seven human proteins homologous to Sir2 (SIRT1 through SIRT7) for cellular localization, expression profiles, protein deacetylation activity, and effects on human cell lifespan. We found that: 1) three nuclear SIRT proteins (SIRT1, SIRT6, and SIRT7) show different subnuclear localizations: SIRT6 and SIRT7 are associated with heterochromatic regions and nucleoli, respectively, where yeast...

Full Text Available Neural circuit development is an activity-dependent process. This activity can be spontaneous, such as the retinal waves that course across the mammalian embryonic retina, or it can be sensory-driven, such as the activation of retinal ganglion cells by visual stimuli. Whichever the source, neural activity provides essential instruction to the developing circuit. Indeed, experimentally altering activity has been shown to impact circuit development and function in many different ways and in many different model systems. In this review we contemplate the idea that retinal waves in amniotes, the animals that develop either in ovo or utero (namely reptiles, birds, mammals could be an evolutionary adaptation to life on land, and that the anamniotes, animals whose development is entirely external (namely the aquatic amphibians and fish, do not display retinal waves, most likely because they simply don’t need them. We then review what is known about the function of both retinal waves and visual stimuli on their respective downstream targets, and predict that the experience-dependent development of the tadpole visual system is a blueprint of what will be found in future studies of the effects of spontaneous retinal waves on instructing development of retinorecipient targets such as the superior colliculus and the lateral geniculate nucleus.

The interactions between PDZ (PSD-95, Dlg, ZO-1) domains and PDZ-binding motifs play central roles in signal transductions within cells. Proteins with PDZ domains bind to PDZ-binding motifs almost exclusively when the motifs are located at the carboxyl (C-) terminal ends of their binding partners. However, it remains little explored whether PDZ-binding motifs show any preferential location at the C-terminal ends of proteins, at genome-level. Here, we examined the distribution of the type-I (x-x-S/T-x-I/L/V) or type-II (x-x-V-x-I/V) PDZ-binding motifs in proteins encoded in the genomes of five different species (human, mouse, zebrafish, fruit fly and nematode). We first established that these PDZ-binding motifs are indeed preferentially present at their C-terminal ends. Moreover, we found specific amino acid (AA) bias for the 'x' positions in the motifs at the C-terminal ends. In general, hydrophilic AAs were favored. Our genomics-based findings confirm and largely extend the results of previous interaction-based studies, allowing us to propose refined consensus sequences for all of the examined PDZ-binding motifs. An ontological analysis revealed that the refined motifs are functionally relevant since a large fraction of the proteins bearing the motif appear to be involved in signal transduction. Furthermore, co-precipitation experiments confirmed two new protein interactions predicted by our genomics-based approach. Finally, we show that influenza virus pathogenicity can be correlated with PDZ-binding motif, with high-virulence viral proteins bearing a refined PDZ-binding motif. Our refined definition of PDZ-binding motifs should provide important clues for identifying functional PDZ-binding motifs and proteins involved in signal transduction.

Full Text Available Abstract Background The interactions between PDZ (PSD-95, Dlg, ZO-1 domains and PDZ-binding motifs play central roles in signal transductions within cells. Proteins with PDZ domains bind to PDZ-binding motifs almost exclusively when the motifs are located at the carboxyl (C- terminal ends of their binding partners. However, it remains little explored whether PDZ-binding motifs show any preferential location at the C-terminal ends of proteins, at genome-level. Results Here, we examined the distribution of the type-I (x-x-S/T-x-I/L/V or type-II (x-x-V-x-I/V PDZ-binding motifs in proteins encoded in the genomes of five different species (human, mouse, zebrafish, fruit fly and nematode. We first established that these PDZ-binding motifs are indeed preferentially present at their C-terminal ends. Moreover, we found specific amino acid (AA bias for the 'x' positions in the motifs at the C-terminal ends. In general, hydrophilic AAs were favored. Our genomics-based findings confirm and largely extend the results of previous interaction-based studies, allowing us to propose refined consensus sequences for all of the examined PDZ-binding motifs. An ontological analysis revealed that the refined motifs are functionally relevant since a large fraction of the proteins bearing the motif appear to be involved in signal transduction. Furthermore, co-precipitation experiments confirmed two new protein interactions predicted by our genomics-based approach. Finally, we show that influenza virus pathogenicity can be correlated with PDZ-binding motif, with high-virulence viral proteins bearing a refined PDZ-binding motif. Conclusions Our refined definition of PDZ-binding motifs should provide important clues for identifying functional PDZ-binding motifs and proteins involved in signal transduction.

The evolutionarilyconserved Hippo inhibitory pathway plays critical roles in tissue homeostasis and organ size control, while mutations affecting certain core components contribute to tumorigenesis. Here we demonstrate that proliferation of Hippo pathway mutant human tumor cells exhibiting high constitutive TEAD transcriptional activity was markedly inhibited by dominant negative TEAD4, which did not inhibit the growth of Hippo wild-type cells with low levels of regulatable TEAD-mediated transcription. The tankyrase inhibitor, XAV939, identified in a screen for inhibitors of TEAD transcriptional activity, phenocopied these effects independently of its other known functions by stabilizing angiomotin and sequestering YAP in the cytosol. We also identified one intrinsically XAV939 resistant Hippo mutant tumor line exhibiting lower and less durable angiomotin stabilization. Thus, angiomotin stabilization provides a new mechanism for targeting tumors with mutations in Hippo pathway core components as well as a biomarker for sensitivity to such therapy.

Identifying conserved alternative splicing (AS) events among evolutionarily distant species can prioritize AS events for functional characterization and help uncover relevant cis- and trans-regulatory factors. A genome-wide search for conserved cassette exon AS events in higher plants revealed the exonization of 5S ribosomal RNA (5S rRNA) within the gene of its own transcription regulator, TFIIIA (transcription factor for polymerase III A). The 5S rRNA-derived exon in TFIIIA gene exists in all representative land plant species but not in green algae and nonplant species, suggesting it is specific to land plants. TFIIIA is essential for RNA polymerase III-based transcription of 5S rRNA in eukaryotes. Integrating comparative genomics and molecular biology revealed that the conserved cassette exon derived from 5S rRNA is coupled with nonsense-mediated mRNA decay. Utilizing multiple independent Arabidopsis overexpressing TFIIIA transgenic lines under osmotic and salt stress, strong accordance between phenotypic and molecular evidence reveals the biological relevance of AS of the exonized 5S rRNA in quantitative autoregulation of TFIIIA homeostasis. Most significantly, this study provides the first evidence of ancient exaptation of 5S rRNA in plants, suggesting a novel gene regulation model mediated by the AS of an anciently exonized noncoding element.

Viral release strategies can be roughly classified as lytic (the ones that accumulate inside the host cell and exit in a burst, killing the cell), and budding (the ones that are produced and released from the host cell gradually). Here we study the evolutionary competition between the two strategies. If all the parameters, such as the rate of viral production, cell life-span and the neutralizing capacity of the antibodies, were the same for lytic and budding viruses, the budding life-strategy would have a large evolutionary advantage. The question arises what makes lytic viruses evolutionarily competitive. We propose that it is the different removal capacity of the antibodies against budding and lytic virions. The latter exit the cell in a large burst such that the antibodies are "flooded" and a larger proportion of virions can escape the immune system and spread to new cells. We create two spatial models of virus-antibody interaction and show that for realistic parameter values, the effect of antibody flooding can indeed take place. We also argue that the lytic life cycle, including a relatively large burst-size, has evolved to promote survival in the face of antibody attack. According to the calculations, in the absence of efficient antibodies, the optimal burst size of lytic viruses would be only a few virus particles, as opposed to the observed 10(2)-10(5) viral particles. Similarly, there is an evolutionary pressure to extend the life-span as a response to antibody action.

Full Text Available Abstract Background Plant HD-Zip transcription factors are modular proteins in which a homeodomain is associated to a leucine zipper. Of the four subfamilies in which they are divided, the tested members from subfamily I bind in vitro the same pseudopalindromic sequence CAAT(A/TATTG and among them, several exhibit similar expression patterns. However, most experiments in which HD-Zip I proteins were over or ectopically expressed under the control of the constitutive promoter 35S CaMV resulted in transgenic plants with clearly different phenotypes. Aiming to elucidate the structural mechanisms underlying such observation and taking advantage of the increasing information in databases of sequences from diverse plant species, an in silico analysis was performed. In addition, some of the results were also experimentally supported. Results A phylogenetic tree of 178 HD-Zip I proteins together with the sequence conservation presented outside the HD-Zip domains allowed the distinction of six groups of proteins. A motif-discovery approach enabled the recognition of an activation domain in the carboxy-terminal regions (CTRs and some putative regulatory mechanisms acting in the amino-terminal regions (NTRs and CTRs involving sumoylation and phosphorylation. A yeast one-hybrid experiment demonstrated that the activation activity of ATHB1, a member of one of the groups, is located in its CTR. Chimerical constructs were performed combining the HD-Zip domain of one member with the CTR of another and transgenic plants were obtained with these constructs. The phenotype of the chimerical transgenic plants was similar to the observed in transgenic plants bearing the CTR of the donor protein, revealing the importance of this module inside the whole protein. Conclusions The bioinformatical results and the experiments conducted in yeast and transgenic plants strongly suggest that the previously poorly analyzed NTRs and CTRs of HD-Zip I proteins play an important

Full Text Available Expression of the MADS domain transcription factor Myocyte Enhancer Factor 2 (MEF2 is regulated by numerous and overlapping enhancers which tightly control its transcription in the mesoderm. To understand how Mef2 expression is controlled in the heart, we identified a late stage Mef2 cardiac enhancer that is active in all heart cells beginning at stage 14 of embryonic development. This enhancer is regulated by the NK-homeodomain transcription factor Tinman, and the GATA transcription factor Pannier through both direct and indirect interactions with the enhancer. Since Tinman, Pannier and MEF2 are evolutionarilyconserved from Drosophila to vertebrates, and since their vertebrate homologs can convert mouse fibroblast cells to cardiomyocytes in different activator cocktails, we tested whether over-expression of these three factors in vivo could ectopically activate known cardiac marker genes. We found that mesodermal over-expression of Tinman and Pannier resulted in approximately 20% of embryos with ectopic Hand and Sulphonylurea receptor (Sur expression. By adding MEF2 alongside Tinman and Pannier, a dramatic expansion in the expression of Hand and Sur was observed in almost all embryos analyzed. Two additional cardiac markers were also expanded in their expression. Our results demonstrate the ability to initiate ectopic cardiac fate in vivo by the combination of only three members of the conserved Drosophila cardiac transcription network, and provide an opportunity for this genetic model system to be used to dissect the mechanisms of cardiac specification.

Innate immunity against pathogen infection is an evolutionarilyconserved process among multicellular organisms. Arabidopsis SNC1 encodes a Resistance protein that combines attributes of multiple mammalian pattern recognition receptors. Utilizing snc1 as an autoimmune model, we identified a discrete protein complex containing at least three members--MOS4 (Modifier Of snc1, 4), AtCDC5, and PRL1 (Pleiotropic Regulatory Locus 1)--that are all essential for plant innate immunity. AtCDC5 has DNA-binding activity, suggesting that this complex probably regulates defense responses through transcriptional control. Since the complex components along with their interactions are highly conserved from fission yeast to Arabidopsis and human, they may also have a yet-to-be-identified function in mammalian innate immunity.

Medullary motoneurons drive vocalization in many vertebrate lineages including fish, amphibians, birds, and mammals. The developmental history of vocal motoneuron populations in each of these lineages remains largely unknown. The highly conservedtranscription factor Paired-like Homeobox 2b (Phox2b) is presumed to be expressed in all vertebrate hindbrain branchial motoneurons, including laryngeal motoneurons essential for vocalization in humans. We used immunohistochemistry and in situ hybridization to examine Phox2b protein and mRNA expression in caudal hindbrain and rostral spinal cord motoneuron populations in seven species across five chordate classes. Phox2b was present in motoneurons dedicated to sound production in mice and frogs (bullfrog, African clawed frog), but not those in bird (zebra finch) or bony fish (midshipman, channel catfish). Overall, the pattern of caudal medullary motoneuron Phox2b expression was conserved across vertebrates and similar to expression in sea lamprey. These observations suggest that motoneurons dedicated to sound production in vertebrates are not derived from a single developmentally or evolutionarilyconserved progenitor pool.

transcript levels were determined in two different parasite lines, 3D7-Lib and NF54-VAR2CSA, expressing VSA associated with severe malaria in children and pregnant women, respectively. The 3D7-Lib showed high transcript levels of Group A var and neighbouring rif genes, whereas rifA2 was found highly...

The identification and study of evolutionarilyconserved genomic sequences that surround disease-related genes is a valuable tool to gain insight into the functional role of these genes and to better elucidate the pathogenetic mechanisms of disease. We created the DG-CST (Disease Gene Conserved Sequence Tags) database for the identification and detailed annotation of human–mouse conserved genomic sequences that are localized within or in the vicinity of human disease-related genes. CSTs are defined as sequences that show at least 70% identity between human and mouse over a length of at least 100 bp. The database contains CST data relative to over 1088 genes responsible for monogenetic human genetic diseases or involved in the susceptibility to multifactorial/polygenic diseases. DG-CST is accessible via the internet at http://dgcst.ceinge.unina.it/ and may be searched using both simple and complex queries. A graphic browser allows direct visualization of the CSTs and related annotations within the context of the relative gene and its transcripts. PMID:15608249

Full Text Available Steroid hormones act, through their respective nuclear receptors, to regulate target gene expression. Despite their critical role in development, physiology, and disease, however, it is still unclear how these systemic cues are refined into tissue-specific responses. We identified a mutation in the evolutionarilyconserved DEAD box RNA helicase belle/DDX3 that disrupts a subset of responses to the steroid hormone ecdysone during Drosophila melanogaster metamorphosis. We demonstrate that belle directly regulates translation of E74A, an ets transcription factor and critical component of the ecdysone-induced transcriptional cascade. Although E74A mRNA accumulates to abnormally high levels in belle mutant tissues, no E74A protein is detectable, resulting in misregulation of E74A-dependent ecdysone response genes. The accumulation of E74A mRNA in belle mutant salivary glands is a result of auto-regulation, fulfilling a prediction made by Ashburner nearly 40 years ago. In this model, Ashburner postulates that, in addition to regulating secondary response genes, protein products of primary response genes like E74A also inhibit their own ecdysone-induced transcription. Moreover, although ecdysone-triggered transcription of E74A appears to be ubiquitous during metamorphosis, belle-dependent translation of E74A mRNA is spatially restricted. These results demonstrate that translational control plays a critical, and previously unknown, role in refining transcriptional responses to the steroid hormone ecdysone.

Full Text Available Steroid hormones act, through their respective nuclear receptors, to regulate target gene expression. Despite their critical role in development, physiology, and disease, however, it is still unclear how these systemic cues are refined into tissue-specific responses. We identified a mutation in the evolutionarilyconserved DEAD box RNA helicase belle/DDX3 that disrupts a subset of responses to the steroid hormone ecdysone during Drosophila melanogaster metamorphosis. We demonstrate that belle directly regulates translation of E74A, an ets transcription factor and critical component of the ecdysone-induced transcriptional cascade. Although E74A mRNA accumulates to abnormally high levels in belle mutant tissues, no E74A protein is detectable, resulting in misregulation of E74A-dependent ecdysone response genes. The accumulation of E74A mRNA in belle mutant salivary glands is a result of auto-regulation, fulfilling a prediction made by Ashburner nearly 40 years ago. In this model, Ashburner postulates that, in addition to regulating secondary response genes, protein products of primary response genes like E74A also inhibit their own ecdysone-induced transcription. Moreover, although ecdysone-triggered transcription of E74A appears to be ubiquitous during metamorphosis, belle-dependent translation of E74A mRNA is spatially restricted. These results demonstrate that translational control plays a critical, and previously unknown, role in refining transcriptional responses to the steroid hormone ecdysone.

Wounds in Drosophila and mouse embryos induce similar genetic pathways to repair epidermal barriers. However, the transcription factors that transduce wound signals to repair epidermal barriers are largely unknown. We characterize the transcriptional regulatory enhancers of 4 genes-Ddc, ple, msn, and kkv-that are rapidly activated in epidermal cells surrounding wounds in late Drosophila embryos and early larvae. These epidermal wound enhancers all contain evolutionarilyconserved sequences matching binding sites for JUN/FOS and GRH transcription factors, but vary widely in trans- and cis-requirements for these inputs and their binding sites. We propose that the combination of GRH and FOS is part of an ancient wound-response pathway still used in vertebrates and invertebrates, but that other mechanisms have evolved that result in similar transcriptional output. A common, but largely untested assumption of bioinformatic analyses of gene regulatory networks is that transcription units activated in the same spatial and temporal patterns will require the same cis-regulatory codes. Our results indicate that this is an overly simplistic view.

Plants are sessile and as such their reactions to environmental challenges differ from those of mobile organisms. Many adaptions involve growth responses and hence, growth regulation is one of the most crucial biological processes for plant survival and fitness. The plant-specific TEOSINTE BRANCHED 1, CYCLOIDEA, PCF1 (TCP) transcription factor family is involved in plant development from cradle to grave, i.e., from seed germination throughout vegetative development until the formation of flowers and fruits. TCP transcription factors have an evolutionary conserved role as regulators in a variety of plant species, including orchids, tomatoes, peas, poplar, cotton, rice and the model plant Arabidopsis. Early TCP research focused on the regulatory functions of TCPs in the development of diverse organs via the cell cycle. Later research uncovered that TCP transcription factors are not static developmental regulators but crucial growth regulators that translate diverse endogenous and environmental signals into growth responses best fitted to ensure plant fitness and health. I will recapitulate the research on TCPs in this review focusing on two topics: the discovery of TCPs and the elucidation of their evolutionarilyconserved roles across the plant kingdom, and the variety of signals, both endogenous (circadian clock, plant hormones) and environmental (pathogens, light, nutrients), TCPs respond to in the course of their developmental roles. PMID:28066483

Plants are sessile and as such their reactions to environmental challenges differ from those of mobile organisms. Many adaptions involve growth responses and hence, growth regulation is one of the most crucial biological processes for plant survival and fitness. The plant-specific TEOSINTE BRANCHED 1, CYCLOIDEA, PCF1 (TCP) transcription factor family is involved in plant development from cradle to grave, i.e., from seed germination throughout vegetative development until the formation of flowers and fruits. TCP transcription factors have an evolutionary conserved role as regulators in a variety of plant species, including orchids, tomatoes, peas, poplar, cotton, rice and the model plant Arabidopsis. Early TCP research focused on the regulatory functions of TCPs in the development of diverse organs via the cell cycle. Later research uncovered that TCP transcription factors are not static developmental regulators but crucial growth regulators that translate diverse endogenous and environmental signals into growth responses best fitted to ensure plant fitness and health. I will recapitulate the research on TCPs in this review focusing on two topics: the discovery of TCPs and the elucidation of their evolutionarilyconserved roles across the plant kingdom, and the variety of signals, both endogenous (circadian clock, plant hormones) and environmental (pathogens, light, nutrients), TCPs respond to in the course of their developmental roles.

TEA domain (TEAD) transcription factors bind to the coactivators YAP and TAZ and regulate the transcriptional output of the Hippo pathway, playing critical roles in organ size control and tumorigenesis. Protein S-palmitoylation attaches a fatty acid, palmitate, to cysteine residues and regulates protein trafficking, membrane localization and signaling activities. Using activity-based chemical probes, we discovered that human TEADs possess intrinsic palmitoylating enzyme-like activities and undergo autopalmitoylation at evolutionarilyconserved cysteine residues under physiological conditions. We determined the crystal structures of lipid-bound TEADs and found that the lipid chain of palmitate inserts into a conserved deep hydrophobic pocket. Strikingly, palmitoylation did not alter TEAD's localization, but it was required for TEAD's binding to YAP and TAZ and was dispensable for its binding to the Vgll4 tumor suppressor. Moreover, palmitoylation-deficient TEAD mutants impaired TAZ-mediated muscle differentiation in vitro and tissue overgrowth mediated by the Drosophila YAP homolog Yorkie in vivo. Our study directly links autopalmitoylation to the transcriptional regulation of the Hippo pathway.

Full Text Available Abstract Background SIN3 is a transcriptional repressor protein known to regulate many genes, including a number of those that encode mitochondrial components. Results By monitoring RNA levels, we find that loss of SIN3 in Drosophila cultured cells results in up-regulation of not only nuclear encoded mitochondrial genes, but also those encoded by the mitochondrial genome. The up-regulation of gene expression is accompanied by a perturbation in ATP levels in SIN3-deficient cells, suggesting that the changes in mitochondrial gene expression result in altered mitochondrial activity. In support of the hypothesis that SIN3 is necessary for normal mitochondrial function, yeast sin3 null mutants exhibit very poor growth on non-fermentable carbon sources and show lower levels of ATP and reduced respiration rates. Conclusions The findings that both yeast and Drosophila SIN3 affect mitochondrial activity suggest an evolutionarilyconserved role for SIN3 in the control of cellular energy production.

FoxA transcription factors play vital roles in regulating the expression of organ-specific genes. BmSGF1, the sole FoxA family member in Bombyx mori, is required for development of the silk gland. However, the function of BmSGF1 in development of the nervous system in the silkworm remains unknown. Here, we show that the amino acids sequence of BmSGF1 is evolutionarilyconserved in its middle region from Trichoplax adhaerens to human and diverged from the homologues in most other species in its N-terminal region. BmSGF1 expresses in the nervous system at the embryonic stage. Knockdown of Bmsgf1 by RNA interference (RNAi) results in abnormal development of axons. Therefore, our results demonstrate that BmSGF1 is an indispensable regulator for neurodevelopment.

The forkhead O (FoxO) family of transcription factors participates in diverse physiologic processes, including induction of cell-cycle arrest, stress resistance, differentiation, apoptosis, and metabolism. Several recent studies indicate that FoxO-dependent signaling is required for long-term regenerative potential of the hematopoietic stem cell (HSC) compartment through regulation of HSC response to physiologic oxidative stress, quiescence, and survival. These observations link FoxO function in mammalian systems with the evolutionarilyconserved role of FoxO in promotion of stress resistance and longevity in lower phylogenetic systems. Furthermore, these findings have implications for aging in higher organisms and in malignant stem cell biology, and suggest that FoxOs may play an important role in the maintenance and integrity of stem cell compartments in a broad spectrum of tissues.

Cathelicidins form a family of vertebrate-specific immune molecules with an evolutionarilyconserved gene structure. We analyzed the expression patterns of cathelicidin genes (CAMP, CATH3, and CATHB1) in chicken bone marrow cells (BMCs) and chicken embryonic fibroblasts (CEFs). We found that CAMP and CATHB1 were significantly up-regulated in BMCs, whereas the expression of CATH3 did not differ significantly between BMCs and CEFs. To study the mechanism underlying the up-regulation of cathelicidin genes in BMCs, we predicted the transcription factors (TFs) that bind to the 5'-flanking regions of cathelicidin genes. CEBPA, EBF1, HES1, MSX1, and ZIC3 were up-regulated in BMCs compared to CEFs. Subsequently, when a siRNA-mediated knockdown assay was performed for MSX1, the expression of CAMP and CATHB1 was decreased in BMCs. We also showed that the transcriptional activity of the CAMP promoter was decreased by mutation of the MSX1-binding sites present within the 5'-flanking region of CAMP. These results increase our understanding of the regulatory mechanisms controlling cathelicidin genes in BMCs.

Plant-defense responses are triggered by perception of conserved microbe-associated molecular patterns (MAMPs), for example, flagellin or peptidoglycan. However, it remained unknown whether plants can detect conserved molecular patterns derived from plant-parasitic animals, including nematodes. Here we show that several genera of plant-parasitic nematodes produce small molecules called ascarosides, an evolutionarilyconserved family of nematode pheromones. Picomolar to micromolar concentratio...

Alternative polyadenylation (APA) is an evolutionarilyconserved mechanism for regulating gene expression. Transcript 3′ end shortening through changes in polyadenylation site usage occurs following T cell activation, but the consequences of APA on gene expression are poorly understood. We previously showed that GU-rich elements (GREs) found in the 3′ untranslated regions of select transcripts mediate rapid mRNA decay by recruiting the protein CELF1/CUGBP1. Using a global RNA sequencing approach, we found that a network of CELF1 target transcripts involved in cell division underwent preferential 3′ end shortening via APA following T cell activation, resulting in decreased inclusion of CELF1 binding sites and increased transcript expression. We present a model whereby CELF1 regulates APA site selection following T cell activation through reversible binding to nearby GRE sequences. These findings provide insight into the role of APA in controlling cellular proliferation during biological processes such as development, oncogenesis and T cell activation PMID:25123787

Transcriptional timing is inherently influenced by gene length, thus providing a mechanism for temporal regulation of gene expression. While gene size has been shown to be important for the expression timing of specific genes during early development, whether it plays a role in the timing of other global gene expression programs has not been extensively explored. Here, we investigate the role of gene length during the early transcriptional response of human fibroblasts to serum stimulation. Using the nascent sequencing techniques Bru-seq and BruUV-seq, we identified immediate genome-wide transcriptional changes following serum stimulation that were linked to rapid activation of enhancer elements. We identified 873 significantly induced and 209 significantly repressed genes. Variations in gene size allowed for a large group of genes to be simultaneously activated but produce full-length RNAs at different times. The median length of the group of serum-induced genes was significantly larger than the median length of all expressed genes, housekeeping genes, and serum-repressed genes. These gene length relationships were also observed in corresponding mouse orthologs, suggesting that relative gene size is evolutionarilyconserved. The sizes of transcription factor and microRNA genes immediately induced after serum stimulation varied dramatically, setting up a cascade mechanism for temporal expression arising from a single activation event. The retention and expansion of large intronic sequences during evolution have likely played important roles in fine-tuning the temporal expression of target genes in various cellular response programs.

TEA domain (TEAD) family transcription factors are key regulators in development, tissue homeostasis and cancer progression. TEAD4 acts as a critical downstream effector of the evolutionarilyconserved Hippo signaling pathway. The well-studied oncogenic protein YAP forms a complex with TEAD4 to regulate gene transcription; so does the tumor suppressor VGLL4. Although it is known that TEAD proteins can bind promoter regions of target genes through the TEA domain, the specific and detailed mechanism of DNA recognition by the TEA domain remains partially understood. Here, we report the crystal structure of TEAD4 TEA domain in complex with a muscle-CAT DNA element. The structure revealed extensive interactions between the TEA domain and the DNA duplex involving both the major and minor grooves of DNA helix. The DNA recognition helix, α3 helix, determines the specificity of the TEA domain binding to DNA sequence. Structure-guided biochemical analysis identified two major binding sites on the interface of the TEA domain-DNA complex. Mutation of TEAD4 at either site substantially decreases its occupancy on the promoter region of target genes, and largely impaired YAP-induced TEAD4 transactivation and target gene transcription, leading to inhibition of growth and colony formation of gastric cancer cell HGC-27. Collectively, our work provides a structural basis for understanding the regulatory mechanism of TEAD-mediated gene transcription.

A CA-repeat microsatellite in insulin-like growth factor 1 (IGF1) promoter was associated with interindividual variation of circulating IGF1 level. Previously, we reported that such association was due to variation of haplotype unit in a linkage disequilibrium block composed of microsatellite and single-nucleotide polymorphisms (SNPs), suggesting the presence of an interaction between them. In this study, reporter assays were performed to investigate the regulatory effect and interaction of genetic variants on gene expression. We used an in vitro system to compare the transcriptional activities of haplotypes (rs35767:T>C, the CA-repeat microsatellite, rs5742612:T>C, and rs2288377:T>A) in evolutionarilyconserved region of IGF1 promoter. In haplotype C-T-T, a longer microsatellite had a lower transcriptional activity (17.6 ± 2.4-fold for 17 repeats and 8.3 ± 1.1-fold for 21 repeats), whereas in haplotype T-C-A, such trend could not be observed, as the microsatellite with 21 repeats had the highest transcriptional activity (17.5 ± 2.3-fold). Because the microsatellite and SNPs affected the transcriptional activity of each other, there may be an interaction between them in the regulation of IGF1 expression. For the first time, we demonstrated that a noncoding microsatellite polymorphism could act as a functional unit and interact with SNPs in the regulation of transcription in human genome.

Cognitive defects in autism spectrum disorder (ASD) include socialization and communication: key behavioral capacities that separate humans from other species. Here, we analyze gene expression in the prefrontal cortex of 63 autism patients and control individuals, as well as 62 chimpanzees and macaques, from natal to adult age. We show that among all aberrant expression changes seen in ASD brains, a single aberrant expression pattern overrepresented in genes involved synaptic-related pathways is enriched in nucleotide variants linked to autism. Furthermore, only this pattern contains an excess of developmental expression features unique to humans, thus resulting in the disruption of human-specific developmental programs in autism. Several members of the early growth response (EGR) transcription factor family can be implicated in regulation of this aberrant developmental change. Our study draws a connection between the genetic risk architecture of autism and molecular features of cortical development unique to humans. PMID:27685936

The shortnose sturgeon, Acipenser brevirostrum, oft considered a phylogenetic relic, is listed as an “endangered species threatened with extinction” in the US and “Vulnerable” on the IUCN Red List. Effective conservation of A. brevirostrum depends on understanding its diversity and evolutionary processes, yet challenges associated with the polyploid nature of its nuclear genome have heretofore limited population genetic analysis to maternally inherited haploid characters. We developed a suite of polysomic microsatellite DNA markers and characterized a sample of 561 shortnose sturgeon collected from major extant populations along the North American Atlantic coast. The 181 alleles observed at 11 loci were scored as binary loci and the data were subjected to multivariate ordination, Bayesian clustering, hierarchical partitioning of variance, and among-population distance metric tests. The methods uncovered moderately high levels of gene diversity suggesting population structuring across and within three metapopulations (Northeast, Mid-Atlantic, and Southeast) that encompass seven demographically discrete and evolutionarily distinct lineages. The predicted groups are consistent with previously described behavioral patterns, especially dispersal and migration, supporting the interpretation that A. brevirostrum exhibit adaptive differences based on watershed. Combined with results of prior genetic (mitochondrial DNA) and behavioral studies, the current work suggests that dispersal is an important factor in maintaining genetic diversity in A. brevirostrum and that the basic unit for conservation management is arguably the local population.

The genes encoding the closely related zinc finger transcription factors Buttonhead (Btd) and D-Sp1 are expressed in the developing limb primordia of Drosophila melanogaster and are required for normal growth of the legs. The D-Sp1 homolog of the red flour beetle Tribolium castaneum, Sp8 (appropriately termed Sp8/9), is also required for the proper growth of the leg segments. Here we report on the isolation and functional study of the Sp8/9 gene from the milkweed bug Oncopeltus fasciatus. We ...

Full Text Available Abstract Background Information about the composition of regulatory regions is of great value for designing experiments to functionally characterize gene expression. The multiplicity of available applications to predict transcription factor binding sites in a particular locus contrasts with the substantial computational expertise that is demanded to manipulate them, which may constitute a potential barrier for the experimental community. Results CBS (Conserved regulatory Binding Sites, http://compfly.bio.ub.es/CBS is a public platform of evolutionarilyconserved binding sites and enhancers predicted in multiple Drosophila genomes that is furnished with published chromatin signatures associated to transcriptionally active regions and other experimental sources of information. The rapid access to this novel body of knowledge through a user-friendly web interface enables non-expert users to identify the binding sequences available for any particular gene, transcription factor, or genome region. Conclusions The CBS platform is a powerful resource that provides tools for data mining individual sequences and groups of co-expressed genes with epigenomics information to conduct regulatory screenings in Drosophila.

Small RNAs are principal elements of bacterial gene regulation and physiology. Two small RNAs in Brucella abortus, AbcR1 and AbcR2, are required for wild-type virulence. Examination of the abcR loci revealed the presence of a gene encoding a LysR-type transcriptional regulator flanking abcR2 on chromosome 1. Deletion of this lysR gene (bab1_1517) resulted in the complete loss of abcR2 expression while no difference in abcR1 expression was observed. The B. abortus bab1_1517 mutant strain was significantly attenuated in macrophages and mice, and bab1_1517 was subsequently named vtlR for virulence-associated transcriptional LysR-family regulator. Microarray analysis revealed three additional genes encoding small hypothetical proteins also under the control of VtlR. Electrophoretic mobility shift assays demonstrated that VtlR binds directly to the promoter regions of abcR2 and the three hypothetical protein-encoding genes, and DNase I footprint analysis identified the specific nucleotide sequence in these promoters that VtlR binds to and drives gene expression. Strikingly, orthologs of VtlR are encoded in a wide range of host-associated α-proteobacteria, and it is likely that the VtlR genetic system represents a common regulatory circuit critical for host-bacterium interactions.

Two genes were identified in rainbow trout that display high sequence identity to vertebrate Lck. Both of the trout Lck transcripts are associated with lymphoid tissues and were found to be highly expressed in IgM-negative lymphocytes. In vitro analysis of trout lymphocytes indicates that trout Lck mRNA is up-regulated by T-cell mitogens, supporting an evolutionarilyconserved function for Lck in the signaling pathways of T-lymphocytes. Here, we describe the generation and characterization of a specific monoclonal antibody raised against the N-terminal domains of recombinant trout Lck that can recognize Lck protein(s) from trout thymocyte lysates that are similar in size (???57 kDa) to mammalian Lck. This antibody also reacted with permeabilized lymphocytes during FACS analysis, indicating its potential usage for cellular analyses of trout lymphocytes, thus representing an important tool for investigations of salmonid T-cell function.

Two homeodomain proteins, Yox1 and Yhp1, act as repressors at early cell cycle boxes (ECBs) to restrict their activity to the M/G1 phase of the cell cycle in budding yeast. These proteins bind to Mcm1 and to a typical homeodomain binding site. The expression of Yox1 is periodic and directly correlated with its binding to, and repression of, ECB activity. The absence of Yox1 and Yhp1 or the constitutive expression of Yox1 leads to the loss of cell-cycle regulation of ECB activity. Therefore, the cell-cycle-regulated expression of these repressors defines the interval of ECB-dependent transcription. Twenty-eight genes, including MCM2-7, CDC6, SWI4, CLN3, and a number of genes required during late M phase have been identified that are coordinately regulated by this pathway.

In animal systems, master regulatory transcription factors (TFs) mediate stem cell maintenance through a direct transcriptional repression of differentiation promoting TFs. Whether similar mechanisms operate in plants is not known. In plants, shoot apical meristems serve as reservoirs of stem cells that provide cells for all above ground organs. WUSCHEL, a homeodomain TF produced in cells of the niche, migrates into adjacent cells where it specifies stem cells. Through high-resolution genomic analysis, we show that WUSCHEL represses a large number of genes that are expressed in differentiating cells including a group of differentiation promoting TFs involved in leaf development. We show that WUS directly binds to the regulatory regions of differentiation promoting TFs; KANADI1, KANADI2, ASYMMETRICLEAVES2 and YABBY3 to repress their expression. Predictions from a computational model, supported by live imaging, reveal that WUS-mediated repression prevents premature differentiation of stem cell progenitors, being part of a minimal regulatory network for meristem maintenance. Our work shows that direct transcriptional repression of differentiation promoting TFs is an evolutionarilyconserved logic for stem cell regulation.

Identification of transcriptional regulatory regions and tracing their internal organization are important for understanding the eukaryotic cell machinery. Cis-regulatory modules (CRMs) of higher eukaryotes are believed to possess a regulatory 'grammar', or preferred arrangement of binding sites, that is crucial for proper regulation and thus tends to be evolutionarilyconserved. Here, we present a method CORECLUST (COnservative REgulatory CLUster STructure) that predicts CRMs based on a set of positional weight matrices. Given regulatory regions of orthologous and/or co-regulated genes, CORECLUST constructs a CRM model by revealing the conserved rules that describe the relative location of binding sites. The constructed model may be consequently used for the genome-wide prediction of similar CRMs, and thus detection of co-regulated genes, and for the investigation of the regulatory grammar of the system. Compared with related methods, CORECLUST shows better performance at identification of CRMs conferring muscle-specific gene expression in vertebrates and early-developmental CRMs in Drosophila.

Summary Background The class V POU domain transcription factor Oct4 (Pou5f1) is a pivotal regulator of embryonic stem cell (ESC) self-renewal and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. Oct4 is also an important evolutionarilyconserved regulator of progenitor cell differentiation during embryonic development. Results Here we examine the function of Oct4 homologs in Xenopus embryos and compare this to the role of Oct4 in maintaining mammalian embryo-derived stem cells. Based on a combination of expression profiling of Oct4/POUV-depleted Xenopus embryos and in silico analysis of existing mammalian Oct4 target data sets, we defined a set of evolutionary-conserved Oct4/POUV targets. Most of these targets were regulators of cell adhesion. This is consistent with Oct4/POUV phenotypes observed in the adherens junctions in Xenopus ectoderm, mouse embryonic, and epiblast stem cells. A number of these targets could rescue both Oct4/POUV phenotypes in cellular adhesion and multipotent progenitor cell maintenance, whereas expression of cadherins on their own could only transiently support adhesion and block differentiation in both ESC and Xenopus embryos. Conclusions Currently, the list of Oct4 transcriptional targets contains thousands of genes. Using evolutionary conservation, we identified a core set of functionally relevant factors that linked the maintenance of adhesion to Oct4/POUV. We found that the regulation of adhesion by the Oct4/POUV network occurred at both transcriptional and posttranslational levels and was required for pluripotency. PMID:24210613

YY2 was originally identified due to its unusual similarity to the evolutionarily well conserved, zinc-finger gene YY1. In this study, we have determined the evolutionary origin and conservation of YY2 using comparative genomic approaches. Our results indicate that YY2 is a retroposed copy of YY1 that has been inserted into another gene locus named Mbtps2 (membrane-bound transcription factor protease site 2). This retroposition is estimated to have occurred after the divergence of placental mammals from other vertebrates based on the detection of YY2 only in the placental mammals. The N-terminal and C-terminal regions of YY2 have evolved under different selection pressures. The N-terminal region has evolved at a very fast pace with very limited functional constraints whereas the DNA-binding, C-terminal region still maintains very similar sequence structure as YY1 and is also well conserved among placental mammals. In situ hybridizations using different adult mouse tissues indicate that mouse YY2 is expressed at relatively low levels in Purkinje and granular cells of cerebellum, and neuronal cells of cerebrum, but at very high levels in testis. The expression levels of YY2 is much lower than YY1, but the overall spatial expression patterns are similar to those of Mbtps2, suggesting a possible shared transcriptional control between YY2 and Mbtps2. Taken together, the formation and evolution of YY2 represent a very unusual case where a transcription factor was first retroposed into another gene locus encoding a protease and survived with different selection schemes and expression patterns.

In our effort to characterize a gene at chromosome 11q13 involved in a t(11;17)(q13;q21) translocation in B-non-Hodgkin lymphoma, we have identified a novel human gene, NOF (Neighbour of FAU). It maps right next to FAU in a head to head configuration separated by a maximum of 146 nucleotides. cDNA clones representing NOF hybridized to a 2. 2-kb mRNA present in all tissues tested. The largest open reading frame appeared to contain 166 amino acids and is proline rich, and the sequence shows no homology with any known gene in the public databases. The NOF gene consists of 4 exons and 3 introns spanning approximately 5 kb, and the boundaries between exons and introns follow the GT/AG rule. The NOF locus is conserved during evolution, with the predicted protein having over 80% identity to three translated mouse and rat ESTs of unknown function. Moreover, the mouse ESTs map in the same organization, closely linked to the FAU gene, in the mouse genome. NOF, however, is not affected by the t(11;17)(q13;q21) chromosomal translocation.

Cytoplasmic polyadenylylation is an evolutionarilyconserved mechanism involved in the translational activation of a set of maternal messenger RNAs (mRNAs) during early development. In this report, we show by interspecies injections that Xenopus and mouse use the same regulatory sequences to control cytoplasmic poly(A) addition during meiotic maturation. Similarly, Xenopus and Drosophila embryos exploit functionally conserved signals to regulate polyadenylylation during early post-fertilizati...

ABA-driven stomatal regulation reportedly evolved after the divergence of ferns, during the early evolution of seed plants approximately 360 Mya. This hypothesis is based on the observation that the stomata of certain fern species are unresponsive to ABA, but exhibit passive hydraulic control. However, ABA-induced stomatal closure was detected in some mosses and lycophytes. Here, we observed that a number of ABA signaling and membrane transporter protein families diversified over the evolutionary history of land plants. The aquatic ferns Azolla filiculoides and Salvinia cucullata have representatives of 23 families of proteins orthologous to those of Arabidopsis thaliana and all other land plant species studied. Phylogenetic analysis of the key ABA signaling proteins indicates an evolutionarilyconserved stomatal response to ABA. Moreover, comparative transcriptomic analysis has identified a suite of ABA responsive genes that differentially expressed in a terrestrial fern species, Polystichum proliferum. These genes encode proteins associated with ABA biosynthesis, transport, reception, transcription, signaling, and ion and sugar transport, which fit the general ABA signaling pathway constructed from Arabidopsis thaliana and Hordeum vulgare. The retention of these key ABA-responsive genes could have had a profound effect on the adaptation of ferns to dry conditions. Furthermore, stomatal assays have shown the primary evidence for ABA-induced closure of stomata in two terrestrial fern species P. proliferum and Nephrolepis exaltata. In summary, we report new molecular and physiological evidence for the presence of active stomatal control in ferns.

Nuclear factor Y (NF-Y) is an evolutionarilyconserved trimeric transcription factor complex present in nearly all eukaryotes. The heterotrimeric NF-Y complex consists of three subunits, NF-YA, NF-YB, and NF-YC, and binds to the CCAAT box in the promoter regions of its target genes to regulate their expression. Yeast and mammal genomes generally have single genes with multiple splicing isoforms that encode each NF-Y subunit. By contrast, plant genomes generally have multi-gene families encoding each subunit and these genes are differentially expressed in various tissues or stages. Therefore, different subunit combinations can lead to a wide variety of NF-Y complexes in various tissues, stages, and growth conditions, indicating the potentially diverse functions of this complex in plants. Indeed, many recent studies have proved that the NF-Y complex plays multiple essential roles in plant growth, development, and stress responses. In this review, we highlight recent progress on NF-Y in Arabidopsis thaliana, including NF-Y protein structure, heterotrimeric complex formation, and the molecular mechanism by which NF-Y regulates downstream target gene expression. We then focus on its biological functions and underlying molecular mechanisms. Finally, possible directions for future research on NF-Y are also presented.

The precise adjustment of the timing of dormancy release according to final grain usage is still a challenge for many cereal crops. Grain sorghum [Sorghum bicolor (L.) Moench] shows wide intraspecific variability in dormancy level and susceptibility to pre-harvest sprouting (PHS). Both embryo sensitivity to abscisic acid (ABA) and gibberellin (GA) metabolism play an important role in the expression of dormancy of the developing sorghum grain. In previous works, it was shown that, simultaneously with a greater embryo sensitivity to ABA and higher expression of SbABA-INSENSITIVE 4 (SbABI4) and SbABA-INSENSITIVE 5 (SbABI5), dormant grains accumulate less active GA4 due to a more active GA catabolism. In this work, it is demonstrated that the ABA signalling components SbABI4 and SbABI5 interact in vitro with a fragment of the SbGA 2-OXIDASE 3 (SbGA2ox3) promoter containing an ABA-responsive complex (ABRC). Both transcription factors were able to bind the promoter, although not simultaneously, suggesting that they might compete for the same cis-acting regulatory sequences. A biological role for these interactions in the expression of dormancy of sorghum grains is proposed: either SbABI4 and/or SbABI5 activate transcription of the SbGA2ox3 gene in vivo and promote SbGA2ox3 protein accumulation; this would result in active degradation of GA4, thus preventing germination of dormant grains. A comparative analysis of the 5'-regulatory region of GA2oxs from both monocots and dicots is also presented; conservation of the ABRC in closely related GA2oxs from Brachypodium distachyon and rice suggest that these species might share the same regulatory mechanism as proposed for grain sorghum.

In evolutionary game theory, evolutionarily stable states are characterised by the folk theorem because exact solutions to the replicator equation are difficult to obtain. It is generally assumed that the folk theorem, which is the fundamental theory for non-cooperative games, defines all Nash equilibria in infinitely repeated games. Here, we prove that Nash equilibria that are not characterised by the folk theorem do exist. By adopting specific reactive strategies, a group of players can be better off by coordinating their actions in repeated games. We call it a type-k equilibrium when a group of k players coordinate their actions and they have no incentive to deviate from their strategies simultaneously. The existence and stability of the type-k equilibrium in general games is discussed. This study shows that the sets of Nash equilibria and evolutionarily stable states have greater cardinality than classic game theory has predicted in many repeated games.

The evolutionarilyconserved family of AP-2 transcription factors (TF) regulates proliferation, differentiation, and apoptosis. Mutations in human AP-2 TF have been linked with bronchio-occular-facial syndrome and Char Syndrome, congenital birth defects characterized by craniofacial deformities and patent ductus arteriosus, respectively. How mutations in AP-2 TF cause the disease phenotypes is not well understood. Here, we characterize the aptf-2(qm27) allele in Caenorhabditis elegans, which carries a point mutation in the conserved DNA binding region of AP-2 TF. We show that compromised APTF-2 activity leads to defects in dorsal intercalation, aberrant ventral enclosure and elongation defects, ultimately culminating in the formation of morphologically deformed larvae or complete arrest during epidermal morphogenesis. Using cell lineaging, we demonstrate that APTF-2 regulates the timing of cell division, primarily in ABarp, D and C cell lineages to control the number of neuroblasts, muscle and epidermal cells. Live imaging revealed nuclear enrichment of APTF-2 in lineages affected by the qm27 mutation preceding the relevant morphogenetic events. Finally, we found that another AP-2 TF, APTF-4, is also essential for epidermal morphogenesis, in a similar yet independent manner. Thus, our study provides novel insight on the cellular-level functions of an AP-2 transcription factor in development. PMID:27176626

Full Text Available Formation of all metazoan bodies is controlled by a group of selector genes including homeobox genes, highly conserved across the entire animal kingdom. The homeobox genes from Pou and Six classes are key members of the regulation cascades determining development of sensory organs, nervous system, gonads and muscles. Besides using common bilaterian models, more attention has recently been targeted at the identification and characterization of these genes within the basal metazoan phyla. Cnidaria as a diploblastic sister group to bilateria with simple and yet specialized organs are suitable models for studies on the sensory organ origin and the associated role of homeobox genes. In this work, Pou and Six homeobox genes, together with a broad range of other sensory-specific transcription factors, were identified in the transcriptome of hydrozoan jellyfish Craspedacusta sowerbyi. Phylogenetic analyses of Pou and Six proteins revealed cnidarian-specific sequence motifs and contributed to the classification of individual factors. The majority of the Craspedacusta sowerbyi Pou and Six homeobox genes are predominantly expressed in statocysts, manubrium and nerve ring, the tissues with sensory and nervous activities. The described diversity and expression patterns of Pou and Six factors in hydrozoan jellyfish highlight their evolutionarilyconserved functions. This study extends the knowledge of the cnidarian genome complexity and shows that the transcriptome of hydrozoan jellyfish is generally rich in homeodomain transcription factors employed in the regulation of sensory and nervous functions.

The mitochondrial DNA control region of the sun bear（Helarctos malayanus） was sequenced using ２１DNA samples collected from confiscated sun bears to identify conservation units, such as evolutionarily significant units and management units, in Sarawak, Borneo Island. A total of １０ haplotypes were observed, indicating the presence of at least two lineages in the sun bear population in Sarawak. Presumably, these two lineages could represent evolutionarily significant units. How...

The developmental mechanisms regulating cell differentiation and patterning during the secondary growth of woody tissues are poorly understood. Class III HD ZIP transcription factors are evolutionarily ancient and play fundamental roles in various aspects of plant development. Here we investigate the role of a Class III HD ZIP transcription factor, ...

Full Text Available Background The classical C2H2 zinc finger domain is involved in a wide range of functions and can bind to DNA, RNA and proteins. The comparison of zinc finger proteins in several eukaryotes has shown that there is a lot of lineage specific diversification and expansion. Although the number of characterized plant proteins that carry the classical C2H2 zinc finger motifs is growing, a systematic classification and analysis of a plant genome zinc finger gene set is lacking. Results We found through in silico analysis 176 zinc finger proteins in Arabidopsis thaliana that hence constitute the most abundant family of putative transcriptional regulators in this plant. Only a minority of 33 A. thaliana zinc finger proteins are conserved in other eukaryotes. In contrast, the majority of these proteins (81% are plant specific. They are derived from extensive duplication events and form expanded families. We assigned the proteins to different subgroups and families and focused specifically on the two largest and evolutionarily youngest families (A1 and C1 that are suggested to be primarily involved in transcriptional regulation. The newly defined family A1 (24 members comprises proteins with tandemly arranged zinc finger domains. Family C1 (64 members, earlier described as the EPF-family in Petunia, comprises proteins with one isolated or two to five dispersed fingers and a mostly invariant QALGGH motif in the zinc finger helices. Based on the amino acid pattern in these helices we could describe five different signature sequences prevalent in C1 zinc finger domains. We also found a number of non-finger domains that are conserved in these families. Conclusions Our analysis of the few evolutionarilyconserved zinc finger proteins of A. thaliana suggests that most of them could be involved in ancient biological processes like RNA metabolism and chromatin-remodeling. In contrast, the majority of the unique A. thaliana zinc finger proteins are known or

Ethylene-responsive element binding factor-associated Amphiphilic Repression (EAR) motif-mediated transcriptional repression is emerging as one of the principal mechanisms of plant gene regulation. The EAR motif, defined by the consensus sequence patterns of either LxLxL or DLNxxP, is the most predominant form of transcriptional repression motif so far identified in plants. Additionally, this active repression motif is highly conserved in transcriptional regulators known to function as negative regulators in a broad range of developmental and physiological processes across evolutionarily diverse plant species. Recent discoveries of co-repressors interacting with EAR motifs, such as TOPLESS (TPL) and AtSAP18, have begun to unravel the mechanisms of EAR motif-mediated repression. The demonstration of genetic interaction between mutants of TPL and AtHDA19, co-complex formation between TPL-related 1 (TPR1) and AtHDA19, as well as direct physical interaction between AtSAP18 and AtHDA19 support a model where EAR repressors, via recruitment of chromatin remodeling factors, facilitate epigenetic regulation of gene expression. Here, we discuss the biological significance of EAR-mediated gene regulation in the broader context of plant biology and present literature evidence in support of a model for EAR motif-mediated repression via the recruitment and action of chromatin modifiers. Additionally, we discuss the possible influences of phosphorylation and ubiquitination on the function and turnover of EAR repressors.

Although a substantial number of hormones and drugs increase cellular cAMP levels, the global impact of cAMP and its major effector mechanism, protein kinase A (PKA), on gene expression is not known. Here we show that treatment of murine wild-type S49 lymphoma cells for 24 h with 8-(4-chlorophenylthio)-cAMP (8-CPTcAMP), a PKA-selective cAMP analog, alters the expression of approx equal to 4,500 of approx. equal to 13,600 unique genes. By contrast, gene expression was unaltered in Kin- S49 cells (that lack PKA) incubated with 8-CPTcAMP. Changes in mRNA and protein expression of several cell cycle regulators accompanied cAMP-induced G1-phase cell-cycle arrest of wild-type S49 cells. Within 2h, 8-CPT-cAMP altered expression of 152 genes that contain evolutionarilyconserved cAMP-response elements within 5 kb of transcriptional start sites, including the circadian clock gene Per1. Thus, cAMP through its activation of PKA produces extensive transcriptional regulation in eukaryotic cells. These transcriptional networks include a primary group of cAMP-response element-containing genes and secondary networks that include the circadian clock.

The C terminus of mammalian transcription factor RAP30 has been found to be a cryptic DNA-binding domain strikingly similar to the C-terminal DNA-binding domain present in conserved region 4 of members of the sigma 70 family of bacterial sigma factors. This RAP30 domain shares strongest sequence similarity with the DNA-binding domain present in region 4 of Bacillus subtilis sporulation-specific sigma K. Like the region 4 DNA-binding activity of Escherichia coli sigma 70, the RAP30 C-terminal DNA binding activity is masked in intact RAP30 but is readily detectable when the RAP30 C terminus is expressed as a fusion protein. Consistent with a role for RAP30 DNA-binding activity in transcription, mutations that abolish DNA binding also abolish transcription. Therefore, RAP30 may function at least in part through the action of an evolutionarily ancient DNA-binding domain that first appeared prior to the divergence of bacteria and eukaryotes.

YchF is an evolutionarilyconserved ATPase of unknown function. In humans, the YchF homologue hOla1 appears to influence cell proliferation and was found to be up-regulated in many tumors. A possible involvement in regulating the oxidative stress response was also suggested, but details on the underlying mechanism are lacking. For gaining insight into YchF function, we used Escherichia coli as a model organism and found that YchF overexpression resulted in H(2)O(2) hypersensitivity. This was not caused by transcriptional or translational down-regulation of H(2)O(2)-scavenging enzymes. Instead, we observed YchF-dependent inhibition of catalase activity and a direct interaction with the major E. coli catalase KatG. KatG inhibition was dependent on the ATPase activity of YchF and was regulated by post-translational modifications, most likely including an H(2)O(2)-dependent dephosphorylation. We furthermore showed that YchF expression is repressed by the transcription factor OxyR and further post-translationally modified in response to H(2)O(2). In summary, our data show that YchF functions as a novel negative regulator of the oxidative stress response in E. coli. Considering the available data on hOla1, YchF/Ola1 most likely execute similar functions in bacteria and humans, and their up-regulation inhibits the ability of the cells to scavenge damaging reactive oxygen species.

In mammals, skin begins as a single-layered epithelium, which, through a series of signals, either stratifies and differentiates to become epidermis or invaginates downward to make hair follicles (HFs). To achieve and maintain proper tissue architecture, keratinocytes must intricately balance growth and differentiation. Here, we uncover a critical and hitherto unappreciated role for Yes-associated protein (YAP), an evolutionarilyconservedtranscriptional coactivator with potent oncogenic potential. We show that YAP is highly expressed and nuclear in single-layered basal epidermal progenitors. Notably, nuclear YAP progressively declines with age and correlates with proliferative potential of epidermal progenitors. Shortly after initiation of HF morphogenesis, YAP translocates to the cytoplasm of differentiating cells. Through genetic analysis, we demonstrate a role for YAP in maintaining basal epidermal progenitors and regulating HF morphogenesis. YAP overexpression causes hair placodes to evaginate into epidermis rather than invaginate into dermis. YAP also expands basal epidermal progenitors, promotes proliferation, and inhibits terminal differentiation. In vitro gain-and-loss of function studies show that primary mouse keratinocytes (MKs) accelerate proliferation, suppress differentiation, and inhibit apoptosis when YAP is activated and reverse these features when YAP is inhibited. Finally, we identify Cyr61 as a target of YAP in MKs and demonstrate a requirement for TEA domain (TEAD) transcriptional factors to comediate YAP functions in MKs.

SWI/SNF ATP-dependent chromatin remodeling complexes (remodelers) perform critical functions in eukaryotic gene expression control. BAP and PBAP are the fly representatives of the two evolutionarilyconserved major subclasses of SWI/SNF remodelers. Both complexes share seven core subunits, including the Brahma ATPase, but differ in a few signature subunits; POLYBROMO and BAP170 specify PBAP, whereas OSA defines BAP. Here, we show that the transcriptional coactivator and PHD finger protein SAYP is a novel PBAP subunit. Biochemical analysis established that SAYP is tightly associated with PBAP but absent from BAP. SAYP, POLYBROMO, and BAP170 display an intimately overlapping distribution on larval salivary gland polytene chromosomes. Genome-wide expression analysis revealed that SAYP is critical for PBAP-dependent transcription. SAYP is required for normal development and interacts genetically with core- and PBAP-selective subunits. Genetic analysis suggested that, like BAP, PBAP also counteracts Polycomb silencing. SAYP appears to be a key architectural component required for the integrity and association of the PBAP-specific module. We conclude that SAYP is a signature subunit that plays a major role in the functional specificity of the PBAP holoenzyme.

Gene expression evolution occurs through changes in cis- or trans-regulatory elements or both. Interactions between transcription factors (TFs) and their binding sites (TFBSs) constitute one of the most important points where these two regulatory components intersect. In this study, we investigated the evolution of TFBSs in the promoter regions of different Saccharomyces strains and species. We divided the promoter of a gene into the proximal region and the distal region, which are defined, respectively, as the 200-bp region upstream of the transcription starting site and as the 200-bp region upstream of the proximal region. We found that the predicted TFBSs in the proximal promoter regions tend to be evolutionarily more conserved than those in the distal promoter regions. Additionally, Saccharomyces cerevisiae strains used in the fermentation of alcoholic drinks have experienced more TFBS losses than gains compared with strains from other environments (wild strains, laboratory strains, and clinical strains). We also showed that differences in TFBSs correlate with the cis component of gene expression evolution between species (comparing S. cerevisiae and its sister species Saccharomyces paradoxus) and within species (comparing two closely related S. cerevisiae strains).

Intimate crosstalk occurs between the pulmonary epithelium and the vascular network during lung development. The transcription factor forkhead box f1 (Foxf1) is expressed in the lung mesenchyme and plays an indispensable role in pulmonary angiogenesis. Sonic hedgehog (Shh), a signalling molecule, is expressed in lung epithelium and is required to establish proper angiogenesis. It has been suggested that Foxf1, a downstream target of the Shh signalling pathway, mediates interaction between angiogenesis and the epithelium in lung. However, there has been no clear evidence showing the mechanism how Foxf1 is regulated by Shh signalling pathway during lung development. In this study, we investigated the lung-specific enhancers of Foxf1 and the Gli binding on the enhancers. At first, we found three evolutionarilyconserved Foxf1 enhancers, two of which were long-range enhancers. Of the long-range enhancers, one demonstrated tissue-specific activity in the proximal and distal pulmonary blood vessels, while the other one demonstrated activity only in distal blood vessels. At analogous positions in human, these long-range enhancers were included in a regulatory region that was reportedly repeatedly deleted in alveolar capillary dysplasia with misalignment of pulmonary vein patients, which indicates the importance of these enhancers in pulmonary blood vessel formation. We also determined that Gli increased the activity of one of these long-range enhancers, which was specific to distal blood vessel, suggesting that Shh regulates Foxf1 transcription in pulmonary distal blood vessel formation.

Developmental transcription factors (DTFs) control development of animals by affecting expression of target genes, some of which are transcription factors themselves. In bilaterians and cnidarians, conserved DTFs are involved in homologous processes such as gastrulation or specification of neurons. The genome of Amphimedon queenslandica, the first sponge to be sequenced, revealed that only a fraction of these conserved DTF families are present in demosponges. This finding was in line with the view that morphological complexity in the animal lineage correlates with developmental toolkit complexity. However, as the phylum Porifera is very diverse, Amphimedon's genome may not be representative of all sponges. The recently sequenced genomes of calcareous sponges Sycon ciliatum and Leucosolenia complicata allowed investigations of DTFs in a sponge lineage evolutionarily distant from demosponges. Surprisingly, the phylogenetic analyses of identified DTFs revealed striking differences between the calcareous sponges and Amphimedon. As these differences appear to be a result of independent gene loss events in the two sponge lineages, the last common ancestor of sponges had to possess a much more diverse repertoire of DTFs than extant sponges. Developmental expression of sponge homologs of genes involved in specification of the Bilaterian endomesoderm and the neurosensory cells suggests that roles of many DTFs date back to the last common ancestor of all animals. Strikingly, even DTFs displaying apparent pan-metazoan conservation of sequence and function are not immune to being lost from individual species genomes. The quest for a comprehensive picture of the developmental toolkit in the last common metazoan ancestor is thus greatly benefitting from the increasing accessibility of sequencing, allowing comparisons of multiple genomes within each phylum.

Harnessing genetic differences between cancerous and noncancerous cells offers a strategy for the development of new therapies. Extrapolating from yeast genetic interaction data, we used cultured human cells and siRNA to construct and evaluate a synthetic lethal interaction network comprised of chromosome instability (CIN) genes that are frequently mutated in colorectal cancer. A small number of genes in this network were found to have synthetic lethal interactions with a large number of canc...

Full Text Available Age-related macular degeneration (AMD is the most common cause of blindness in the elderly population. Hypoxic stress created in the micro-environment of the photoreceptors is thought to be the underlying cause that results in the pathophysiology of AMD. However, association of AMD with alternative splicing mediated gene regulation is not well explored. Alternative Splicing is one of the primary mechanisms in humans by which fewer protein coding genes are able to generate a vast proteome. Here, we investigated the expression of a known stress response gene and an alternative splicing factor called Serine-Arginine rich splicing factor 10 (Sfrs10. Sfrs10 is a member of the serine-arginine (SR rich protein family and is 100% identical at the amino acid level in most mammals. Immunoblot analysis on retinal extracts from mouse, rat, and chicken showed a single immunoreactive band. Further, immunohistochemistry on adult mouse, rat and chicken retinae showed pan-retinal expression. However, SFRS10 was not detected in normal human retina but was observed as distinct nuclear speckles in AMD retinae. This is in agreement with previous reports that show Sfrs10 to be a stress response gene, which is upregulated under hypoxia. The difference in the expression of Sfrs10 between humans and lower mammals and the upregulation of SFRS10 in AMD is further reflected in the divergence of the promoter sequence between these species. Finally, SFRS10+ speckles were independent of the SC35+ SR protein speckles or the HSF1+ stress granules. In all, our data suggests that SFRS10 is upregulated and forms distinct stress-induced speckles and might be involved in AS of stress response genes in AMD.

In plants, algae, and cyanobacteria, photosystem II (PSII) catalyzes the light-driven oxidation of water. The oxygen-evolving complex of PSII is a Mn4CaO5 cluster embedded in a well-defined protein environment in the thylakoid membrane. However, transport of manganese and calcium into the thylakoid...... by oxygen evolution rates) and for manganese incorporation. Manganese binding to PSII was severely reduced in pam71 thylakoids, particularly in PSII supercomplexes. In cation partitioning assays with intact chloroplasts, Mn2+ and Ca2+ ions were differently sequestered in pam71, with Ca2+ enriched in pam71...

In the absence of effective neuroprotective agents in the clinic, ischemic and pharmacological preconditioning are gaining increased interest in the field of cerebral ischemia. Our lab recently reported that resveratrol preconditioning affords tolerance against a focal cerebral ischemic insult in mice that can last for at least 14 days in vivo making it the longest window of ischemic tolerance discovered to date by a single administration of a pharmacological agent. The mechanism behind this novel extended window of ischemic tolerance remains elusive. In the below commentary we discuss potential mechanisms that could explain this novel extended window of ischemic tolerance in the context of previously identified windows and the known mechanisms behind them. We also draw parallels from the fields of hibernation and hypoxia-tolerance, which are chronic adaptations to severe conditions of hypoxia and ischemia known to be mediated by a form of metabolic depression. We also briefly discuss the importance of epigenetic modifications in maintaining this depressed state of metabolism. PMID:27796011

Overexpression of human epidermal growth factor receptor 2 (HER2) is associated with tumor aggressiveness and poor prognosis in breast cancer. With the availability of therapeutic antibodies against HER2, great strides have been made in the clinical management of HER2 overexpressing breast cancer...... for potential therapeutic application. Thus, this novel computational process for predicting or searching for potential epitopes or key target sites may contribute to epitope-based vaccine and function-selected drug design, especially when x-ray crystal structure protein data is not available....

Full Text Available The evolution of heteromorphic sex chromosomes has repeatedly resulted in the evolution of sex chromosome-specific forms of regulation, including sex chromosome dosage compensation in the soma and meiotic sex chromosome inactivation in the germline. In the male germline of Drosophila melanogaster, a novel but poorly understood form of sex chromosome-specific transcriptional regulation occurs that is distinct from canonical sex chromosome dosage compensation or meiotic inactivation. Previous work shows that expression of reporter genes driven by testis-specific promoters is considerably lower-approximately 3-fold or more-for transgenes inserted into X chromosome versus autosome locations. Here we characterize this transcriptional suppression of X-linked genes in the male germline and its evolutionary consequences. Using transgenes and transpositions, we show that most endogenous X-linked genes, not just testis-specific ones, are transcriptionally suppressed several-fold specifically in the Drosophila male germline. In wild-type testes, this sex chromosome-wide transcriptional suppression is generally undetectable, being effectively compensated by the gene-by-gene evolutionary recruitment of strong promoters on the X chromosome. We identify and experimentally validate a promoter element sequence motif that is enriched upstream of the transcription start sites of hundreds of testis-expressed genes; evolutionarilyconserved across species; associated with strong gene expression levels in testes; and overrepresented on the X chromosome. These findings show that the expression of X-linked genes in the Drosophila testes reflects a balance between chromosome-wide epigenetic transcriptional suppression and long-term compensatory adaptation by sex-linked genes. Our results have broad implications for the evolution of gene expression in the Drosophila male germline and for genome evolution.

The evolution of heteromorphic sex chromosomes has repeatedly resulted in the evolution of sex chromosome-specific forms of regulation, including sex chromosome dosage compensation in the soma and meiotic sex chromosome inactivation in the germline. In the male germline of Drosophila melanogaster, a novel but poorly understood form of sex chromosome-specific transcriptional regulation occurs that is distinct from canonical sex chromosome dosage compensation or meiotic inactivation. Previous work shows that expression of reporter genes driven by testis-specific promoters is considerably lower-approximately 3-fold or more-for transgenes inserted into X chromosome versus autosome locations. Here we characterize this transcriptional suppression of X-linked genes in the male germline and its evolutionary consequences. Using transgenes and transpositions, we show that most endogenous X-linked genes, not just testis-specific ones, are transcriptionally suppressed several-fold specifically in the Drosophila male germline. In wild-type testes, this sex chromosome-wide transcriptional suppression is generally undetectable, being effectively compensated by the gene-by-gene evolutionary recruitment of strong promoters on the X chromosome. We identify and experimentally validate a promoter element sequence motif that is enriched upstream of the transcription start sites of hundreds of testis-expressed genes; evolutionarilyconserved across species; associated with strong gene expression levels in testes; and overrepresented on the X chromosome. These findings show that the expression of X-linked genes in the Drosophila testes reflects a balance between chromosome-wide epigenetic transcriptional suppression and long-term compensatory adaptation by sex-linked genes. Our results have broad implications for the evolution of gene expression in the Drosophila male germline and for genome evolution.

Mammalian sex chromosomes evolved from the degeneration of one homolog of a pair of ancestral autosomes, the proto-Y. This resulted in a gene dose imbalance that is believed to be restored (partially or fully) through upregulation of gene expression from the single active X-chromosome in both sexes by a dosage compensatory mechanism. We analyzed multiple genome-wide RNA stability data sets and found significantly longer average half-lives for X-chromosome transcripts than for autosomal transcripts in various human cell lines, both male and female, and in mice. Analysis of ribosome profiling data shows that ribosome density is higher on X-chromosome transcripts than on autosomal transcripts in both humans and mice, suggesting that the higher stability is causally linked to a higher translation rate. Our results and observations are in accordance with a dosage compensatory upregulation of expressed X-linked genes. We therefore propose that differential mRNA stability and translation rates of the autosomes and sex chromosomes contribute to an evolutionarilyconserved dosage compensation mechanism in mammals.

National Oceanic and Atmospheric Administration, Department of Commerce — Data sets used to support analysis published by O'Corry-Crowe et al (2014) Crossing to safety: Dispersal, colonization and mate choice in evolutionarily distinct...

GATA genes are an evolutionarilyconserved family, which encode a group of important transcription factors involved in the regulation of diverse processes including the development of the heart, haematopoietic system and sex gonads. However, the evolutionary history of the GATA family has not been completely understood. We constructed a complete phylogenetic tree with functional domain information of the GATA genes of both vertebrates and several invertebrates, and mapped the GATA genes onto relevant chromosomes. Conserved synteny was observed around the GATA loci on the chromosomes. GATAs have a tendency to segregate onto different chromosomes during evolution. The phylogenetic tree is consistent with the relevant functions of GATA members. Analysis of the zinc finger domain showed that the domain tends to be duplicated during evolution from invertebrates to vertebrates. We propose that the balance between duplications of zinc finger domains and GATA members should be maintained to exert their physiological roles in each evolutionary stage. Therefore, evolutionary pressure on the GATAs must exist to maintain the balance during evolution from invertebrates to vertebrates. These results reveal the evolutionary characteristics of the GATA family and contribute to a better understanding of the relationship between evolution and biological functions of the gene family, which will help to uncover the GATAs’ biological roles, evolution and their relationship with associated diseases.

Full Text Available GATA transcription factors are evolutionarilyconservedtranscriptional regulators that recognize promoter elements with a G-A-T-A core sequence. In comparison to animal genomes, the GATA transcription factor family in plants is comparatively large with approximately 30 members. In spite of a long-standing interest of plant molecular biologists in GATA factors, only research conducted in the last years has led to reliable insights into their functions during plant development. Here, we review the current knowledge on B-GATAs, one of four GATA factor subfamilies from Arabidopsis thaliana. We show that B-GATAs can be subdivided based on structural features and their biological function into family members with a C-terminal LLM- (leucine-leucine-methionine domain or an N-terminal HAN- (HANABA TARANU domain. The paralogous GNC (GATA, NITRATE-INDUCIBLE, CARBON-METABOLISM INVOLVED and CGA1/GNL (CYTOKININ-INDUCED GATA1/GNC-LIKE are introduced as LLM-domain containing B-GATAs from Arabidopsis that control germination, greening, senescence and flowering time downstream from several growth regulatory signals including light and the hormones gibberellin, auxin, and cytokinin. Arabidopsis HAN and its monocot-specific paralogs from rice (NECK LEAF1, maize (TASSEL SHEATH1, and barley (THIRD OUTER GLUME are HAN-domain-containing B-GATAs with a predominant role in embryo development and floral development. We also review GATA23, a regulator of lateral root initiation from Arabidopsis, that is closely related to GNC and GNL but has a degenerate LLM-domain that is seemingly specific for the Brassicaceae family. The Brassicaceae-specific GATA23 together with the above-mentioned monocot-specific HAN-domain GATAs provide evidence that neofunctionalization of the B-GATAs was used during plant evolution to expand the functional repertoire of these transcription factors.

Evolutionary studies benefit from deep sequencing technologies that generate genomic and transcriptomic sequences from a variety of organisms. Genome sequencing and RNAseq have complementary strengths. In this study, we present the assembly of the most complete Hydra transcriptome to date along with a comparative analysis of the specific features of RNAseq and genome-predicted transcriptomes currently available in the freshwater hydrozoan Hydra vulgaris. To produce an accurate and extensive Hydra transcriptome, we combined Illumina and 454 Titanium reads, giving the primacy to Illumina over 454 reads to correct homopolymer errors. This strategy yielded an RNAseq transcriptome that contains 48'909 unique sequences including splice variants, representing approximately 24'450 distinct genes. Comparative analysis to the available genome-predicted transcriptomes identified 10'597 novel Hydra transcripts that encode 529 evolutionarily-conserved proteins. The annotation of 170 human orthologs points to critical functions in protein biosynthesis, FGF and TOR signaling, vesicle transport, immunity, cell cycle regulation, cell death, mitochondrial metabolism, transcription and chromatin regulation. However, a majority of these novel transcripts encodes short ORFs, at least 767 of them corresponding to pseudogenes. This RNAseq transcriptome also lacks 11'270 predicted transcripts that correspond either to silent genes or to genes expressed below the detection level of this study. We established a simple and powerful strategy to combine Illumina and 454 reads and we produced, with genome assistance, an extensive and accurate Hydra transcriptome. The comparative analysis of the RNAseq transcriptome with genome-predicted transcriptomes lead to the identification of large populations of novel as well as missing transcripts that might reflect Hydra-specific evolutionary events.

Interspecific mutualisms are often vulnerable to instability because low benefit : cost ratios can rapidly lead to extinction or to the conversion of mutualism to parasite–host or predator–prey interactions. We hypothesize that the evolutionary stability of mutualism can depend on how benefits and costs to one mutualist vary with the population density of its partner, and that stability can be maintained if a mutualist can influence demographic rates and regulate the population density of its partner. We test this hypothesis in a model of mutualism with key features of senita cactus (Pachycereus schottii) – senita moth (Upiga virescens) interactions, in which benefits of pollination and costs of larval seed consumption to plant fitness depend on pollinator density. We show that plants can maximize their fitness by allocating resources to the production of excess flowers at the expense of fruit. Fruit abortion resulting from excess flower production reduces pre–adult survival of the pollinating seed–consumer, and maintains its density beneath a threshold that would destabilize the mutualism. Such a strategy of excess flower production and fruit abortion is convergent and evolutionarily stable against invasion by cheater plants that produce few flowers and abort few to no fruit. This novel mechanism of achieving evolutionarily stable mutualism, namely interspecific population regulation, is qualitatively different from other mechanisms invoking partner choice or selective rewards, and may be a general process that helps to preserve mutualistic interactions in nature.

The mosquito Anopheles gambiae is an important vector for malaria, which is one of the most serious human parasitic diseases in the world, causing up to 2.7 million deaths yearly. To contribute to our understanding of A. gambiae and to the transmission of malaria, we have now cloned four evolutio......The mosquito Anopheles gambiae is an important vector for malaria, which is one of the most serious human parasitic diseases in the world, causing up to 2.7 million deaths yearly. To contribute to our understanding of A. gambiae and to the transmission of malaria, we have now cloned four...... evolutionarily related G protein-coupled receptors (GPCRs) from this mosquito and expressed them in Chinese hamster ovary cells. After screening of a library of thirty-three insect or other invertebrate neuropeptides and eight biogenic amines, we could identify (de-orphanize) three of these GPCRs as...... relationship to the A. gambiae and other insect AKH receptors suggested that it is a receptor for an AKH-like peptide. This is the first published report on evolutionarily related AKH, corazonin, and CCAP receptors in mosquitoes....

Background MicroRNAs (miRNA) are small (20-25 nt) non-coding RNA molecules that regulate gene expression through interaction with mRNA in plants and metazoans. A few hundred miRNAs are known or predicted, and most of those are evolutionarilyconserved. In general plant miRNA are different from...

textabstractDrosophila BAP and PBAP represent two evolutionarilyconserved subclasses of SWI/SNF chromatin remodelers. The two complexes share the same core subunits, including the BRM ATPase, but differ in a few signature subunits: OSA defines BAP, whereas Polybromo (PB) and BAP170 specify PBAP. He

As in all other eukaryotic organisms, endoplasmic reticulum (ER) stress triggers the evolutionarilyconserved unfolded protein response in soybean, but it also communicates with other adaptive signaling responses, such as osmotic stress-induced and ER stress-induced programmed cell death. These two signaling pathways converge at the level of gene transcription to activate an integrated cascade that is mediated by N-rich proteins (NRPs). Here, we describe a novel transcription factor, GmERD15 (Glycine max Early Responsive to Dehydration 15), which is induced by ER stress and osmotic stress to activate the expression of NRP genes. GmERD15 was isolated because of its capacity to stably associate with the NRP-B promoter in yeast. It specifically binds to a 187-bp fragment of the NRP-B promoter in vitro and activates the transcription of a reporter gene in yeast. Furthermore, GmERD15 was found in both the cytoplasm and the nucleus, and a ChIP assay revealed that it binds to the NRP-B promoter in vivo. Expression of GmERD15 in soybean protoplasts activated the NRP-B promoter and induced expression of the NRP-B gene. Collectively, these results support the interpretation that GmERD15 functions as an upstream component of stress-induced NRP-B-mediated signaling to connect stress in the ER to an osmotic stress-induced cell death signal.

Treatment of diverticulitis comprises at least two options: conservative or surgical management. There is a recent trend to limit surgical treatment of acute diverticulitis and to favor conservative management. This review addresses general aspects of conservative patient care with special focus on the treatment of patients with a first attack of diverticulitis. The presentation does not include a discussion of specific drugs which is given in other sections of this issue.

Full Text Available The production of ribosomes is ubiquitous and fundamental to life. As such, it is surprising that defects in ribosome biogenesis underlie a growing number of symptomatically distinct inherited disorders, collectively called ribosomopathies. We previously determined that the nucleolar protein, NOL11, is essential for optimal pre-rRNA transcription and processing in human tissue culture cells. However, the role of NOL11 in the development of a multicellular organism remains unknown. Here, we reveal a critical function for NOL11 in vertebrate ribosome biogenesis and craniofacial development. Nol11 is strongly expressed in the developing cranial neural crest (CNC of both amphibians and mammals, and knockdown of Xenopus nol11 results in impaired pre-rRNA transcription and processing, increased apoptosis, and abnormal development of the craniofacial cartilages. Inhibition of p53 rescues this skeletal phenotype, but not the underlying ribosome biogenesis defect, demonstrating an evolutionarilyconserved control mechanism through which ribosome-impaired craniofacial cells are removed. Excessive activation of this mechanism impairs craniofacial development. Together, our findings reveal a novel requirement for Nol11 in craniofacial development, present the first frog model of a ribosomopathy, and provide further insight into the clinically important relationship between specific ribosome biogenesis proteins and craniofacial cell survival.

PDX1 is a homeodomain transcription factor essential for pancreatic development and mature beta cell function. Homeodomain proteins typically recognize short TAAT DNA motifs in vitro: this binding displays paradoxically low specificity and affinity, given the extremely high specificity of action of these proteins in vivo. To better understand how PDX1 selects target genes in vivo, we have examined the interaction of PDX1 with natural and artificial binding sites. Comparison of PDX1 binding sites in several target promoters revealed an evolutionarilyconserved pattern of nucleotides flanking the TAAT core. Using competitive in vitro DNA binding assays, we defined three groups of binding sites displaying high, intermediate and low affinity. Transfection experiments revealed a striking correlation between the ability of each sequence to activate transcription in cultured beta cells, and its ability to bind PDX1 in vitro. Site selection from a pool of oligonucleotides (sequence NNNTAATNNN) revealed a non-random preference for particular nucleotides at the flanking locations, resembling natural PDX1 binding sites. Taken together, the data indicate that the intrinsic DNA binding specificity of PDX1, in particular the bases adjacent to TAAT, plays an important role in determining the spectrum of target genes. PMID:14704343

The homeostatic maintenance of the genomic DNA is crucial for regulating aging processes. However, the role of RNA homeostasis in aging processes remains unknown. RNA helicases are a large family of enzymes that regulate the biogenesis and homeostasis of RNA. However, the functional significance of RNA helicases in aging has not been explored. Here, we report that a large fraction of RNA helicases regulate the lifespan of Caenorhabditis elegans. In particular, we show that a DEAD-box RNA helicase, helicase 1 (HEL-1), promotes longevity by specifically activating the DAF-16/forkhead box O (FOXO) transcription factor signaling pathway. We find that HEL-1 is required for the longevity conferred by reduced insulin/insulin-like growth factor 1 (IGF-1) signaling (IIS) and is sufficient for extending lifespan. We further show that the expression of HEL-1 in the intestine and neurons contributes to longevity. HEL-1 enhances the induction of a large fraction of DAF-16 target genes. Thus, the RNA helicase HEL-1 appears to promote longevity in response to decreased IIS as a transcription coregulator of DAF-16. Because HEL-1 and IIS are evolutionarily well conserved, a similar mechanism for longevity regulation via an RNA helicase-dependent regulation of FOXO signaling may operate in mammals, including humans.

Full Text Available Abstract Background ATRX is a tightly-regulated multifunctional protein with crucial roles in mammalian development. Mutations in the ATRX gene cause ATR-X syndrome, an X-linked recessive developmental disorder resulting in severe mental retardation and mild alpha-thalassemia with facial, skeletal and genital abnormalities. Although ubiquitously expressed the clinical features of the syndrome indicate that ATRX is not likely to be a global regulator of gene expression but involved in regulating specific target genes. The regulation of ATRX expression is not well understood and this is reflected by the current lack of identified upstream regulators. The availability of genomic data from a range of species and the very highly conserved 5' regulatory regions of the ATRX gene has allowed us to investigate putative transcription factor binding sites (TFBSs in evolutionarilyconserved regions of the mammalian ATRX promoter. Results We identified 12 highly conserved TFBSs of key gene regulators involved in biologically relevant processes such as neural and testis development and alpha-globin regulation. Conclusions Our results reveal potentially important regulatory elements in the ATRX gene which may lead to the identification of upstream regulators of ATRX and aid in the understanding of the molecular mechanisms that underlie ATR-X syndrome.

Discriminating pathogenic bacteria from bacteria used as a food source is key to Caenorhabidits elegans immunity. Using mutants defective in the enzymes of O-linked N-acetylglucosamine (O-GlcNAc) cycling, we examined the role of this nutrient-sensing pathway in the C. elegans innate immune response. Genetic analysis showed that deletion of O-GlcNAc transferase (ogt-1) yielded animals hypersensitive to the human pathogen S. aureus but not to P. aeruginosa. Genetic interaction studies revealed that nutrient-responsive OGT-1 acts through the conserved β-catenin (BAR-1) pathway and in concert with p38 MAPK (PMK-1) to modulate the immune response to S. aureus. Moreover, whole genome transcriptional profiling revealed that O-GlcNAc cycling mutants exhibited deregulation of unique stress- and immune-responsive genes. The participation of nutrient sensor OGT-1 in an immunity module evolutionarilyconserved from C. elegans to humans reveals an unexplored nexus between nutrient availability and a pathogen-specific immune response.

Seven purified proteins may be combined to reconstitute regulated, promoter-dependent RNA polymerase II transcription: five general transcription factors, Mediator, and RNA polymerase II. The entire system has been conserved across species from yeast to humans. The structure of RNA polymerase II, consisting of 10 polypeptides with a mass of about 500 kDa, has been determined at atomic resolution. On the basis of this structure, that of an actively transcribing RNA polymerase II complex has been determined as well.

The LIM only protein 2 (LMO2) is a key regulator of hematopoietic stem cell development whose ectopic expression in T cells leads to the onset of acute lymphoblastic leukemia. Through its LIM domains, LMO2 is thought to function as the scaffold for a DNA-binding transcription regulator complex, including the basic helix-loop-helix proteins SCL/TAL1 and E47, the zinc finger protein GATA-1, and LIM-domain interacting protein LDB1. To understand the role of LMO2 in the formation of this complex and ultimately to dissect its function in normal and aberrant hematopoiesis, we solved the crystal structure of LMO2 in complex with the LID domain of LDB1 at 2.4 Å resolution. We observe a largely unstructured LMO2 kept in register by the LID binding both LIM domains. Comparison of independently determined crystal structures of LMO2 reveals large movements around a conserved hinge between the LIM domains. We demonstrate that such conformational flexibility is necessary for binding of LMO2 to its partner protein SCL/TAL1 in vitro and for the function of this complex in vivo. These results, together with molecular docking and analysis of evolutionarilyconserved residues, yield the first structural model of the DNA-binding complex containing LMO2, LDB1, SCL/TAL1, and GATA-1.

The plant-specific DNA-dependent RNA polymerase V (Pol V) evolved from Pol II to function in an RNA-directed DNA methylation pathway. Here, we have identified targets of Pol V in Arabidopsis thaliana on a genome-wide scale using ChIP-seq of NRPE1, the largest catalytic subunit of Pol V. We found that Pol V is enriched at promoters and evolutionarily recent transposons. This localization pattern is highly correlated with Pol V-dependent DNA methylation and small RNA accumulation. We also show that genome-wide chromatin association of Pol V is dependent on all members of a putative chromatin-remodeling complex termed DDR. Our study presents a genome-wide view of Pol V occupancy and sheds light on the mechanistic basis of Pol V localization. Furthermore, these findings suggest a role for Pol V and RNA-directed DNA methylation in genome surveillance and in responding to genome evolution.

The cooperative evolutionary stability under self-organised organization is discussed in this paper.The differences between the objects studied by cooperative game theory and the ones studied by cooperative game in science & technology alliance are analyzed.The mutant probability of agent's utility under endogenous technical factor condition is analyzed.By clarifying the connotation of Pareto-dominate institution in cooperative game,the efficient and feasible managerial definition of Pareto-dominate Institution in science & technology alliance is presented.The evolutionarily cooperative game for the agent in Pareto-dominate institution is explained.And then the necessary condition of cooperative evolutionary stabilization based on multi-agent utility's dynamic equilibrium is put forward.Finally,the model of alliance's utility's dynamic equilibrium under self-organization is established.

Full Text Available Abstract Background Accurate computational identification of cis-regulatory motifs is difficult, particularly in eukaryotic promoters, which typically contain multiple short and degenerate DNA sequences bound by several interacting factors. Enrichment in combinations of rare motifs in the promoter sequence of functionally or evolutionarily related genes among several species is an indicator of conservedtranscriptional regulatory mechanisms. This provides a basis for the computational identification of cis-regulatory motifs. Results We have used a discriminative seeding DNA motif discovery algorithm for an in-depth analysis of 54 seed storage protein (SSP gene promoters from three plant families, namely Brassicaceae (mustards, Fabaceae (legumes and Poaceae (grasses using backgrounds based on complete sets of promoters from a representative species in each family, namely Arabidopsis (Arabidopsis thaliana (L. Heynh., soybean (Glycine max (L. Merr. and rice (Oryza sativa L. respectively. We have identified three conserved motifs (two RY-like and one ACGT-like in Brassicaceae and Fabaceae SSP gene promoters that are similar to experimentally characterized seed-specific cis-regulatory elements. Fabaceae SSP gene promoter sequences are also enriched in a novel, seed-specific E2Fb-like motif. Conserved motifs identified in Poaceae SSP gene promoters include a GCN4-like motif, two prolamin-box-like motifs and an Skn-1-like motif. Evidence of the presence of a variant of the TATA-box is found in the SSP gene promoters from the three plant families. Motifs discovered in SSP gene promoters were used to score whole-genome sets of promoters from Arabidopsis, soybean and rice. The highest-scoring promoters are associated with genes coding for different subunits or precursors of seed storage proteins. Conclusion Seed storage protein gene promoter motifs are conserved in diverse species, and different plant families are characterized by a distinct combination

Background Accurate computational identification of cis-regulatory motifs is difficult, particularly in eukaryotic promoters, which typically contain multiple short and degenerate DNA sequences bound by several interacting factors. Enrichment in combinations of rare motifs in the promoter sequence of functionally or evolutionarily related genes among several species is an indicator of conservedtranscriptional regulatory mechanisms. This provides a basis for the computational identification of cis-regulatory motifs. Results We have used a discriminative seeding DNA motif discovery algorithm for an in-depth analysis of 54 seed storage protein (SSP) gene promoters from three plant families, namely Brassicaceae (mustards), Fabaceae (legumes) and Poaceae (grasses) using backgrounds based on complete sets of promoters from a representative species in each family, namely Arabidopsis (Arabidopsis thaliana (L.) Heynh.), soybean (Glycine max (L.) Merr.) and rice (Oryza sativa L.) respectively. We have identified three conserved motifs (two RY-like and one ACGT-like) in Brassicaceae and Fabaceae SSP gene promoters that are similar to experimentally characterized seed-specific cis-regulatory elements. Fabaceae SSP gene promoter sequences are also enriched in a novel, seed-specific E2Fb-like motif. Conserved motifs identified in Poaceae SSP gene promoters include a GCN4-like motif, two prolamin-box-like motifs and an Skn-1-like motif. Evidence of the presence of a variant of the TATA-box is found in the SSP gene promoters from the three plant families. Motifs discovered in SSP gene promoters were used to score whole-genome sets of promoters from Arabidopsis, soybean and rice. The highest-scoring promoters are associated with genes coding for different subunits or precursors of seed storage proteins. Conclusion Seed storage protein gene promoter motifs are conserved in diverse species, and different plant families are characterized by a distinct combination of conserved motifs

Full Text Available Abtract Background The dinoflagellates Durinskia baltica and Kryptoperidinium foliaceum are distinguished by the presence of a tertiary plastid derived from a diatom endosymbiont. The diatom is fully integrated with the host cell cycle and is so altered in structure as to be difficult to recognize it as a diatom, and yet it retains a number of features normally lost in tertiary and secondary endosymbionts, most notably mitochondria. The dinoflagellate host is also reported to retain mitochondrion-like structures, making these cells unique in retaining two evolutionarily distinct mitochondria. This redundancy raises the question of whether the organelles share any functions in common or have distributed functions between them. Results We show that both host and endosymbiont mitochondrial genomes encode genes for electron transport proteins. We have characterized cytochrome c oxidase 1 (cox1, cytochrome oxidase 2 (cox2, cytochrome oxidase 3 (cox3, cytochrome b (cob, and large subunit of ribosomal RNA (LSUrRNA of endosymbiont mitochondrial ancestry, and cox1 and cob of host mitochondrial ancestry. We show that all genes are transcribed and that those ascribed to the host mitochondrial genome are extensively edited at the RNA level, as expected for a dinoflagellate mitochondrion-encoded gene. We also found evidence for extensive recombination in the host mitochondrial genes and that recombination products are also transcribed, as expected for a dinoflagellate. Conclusion Durinskia baltica and K. foliaceum retain two mitochondria from evolutionarily distinct lineages, and the functions of these organelles are at least partially overlapping, since both express genes for proteins in electron transport.

Heat shock transcription factor (HSF), an evolutionarilyconserved stress response regulator, forms trimers and binds to heat shock element (HSE), comprising at least three continuous inverted repeats of the sequence 5'-nGAAn-3'. The single HSF of yeast is also able to bind discontinuously arranged nGAAn units. We investigated interactions between three human HSFs and various HSE types in vitro, in yeast cells, and in HeLa cells. Human HSF1, a stress-activated regulator, preferentially bound to continuous HSEs rather than discontinuous HSEs, and heat shock of HeLa cells caused expression of reporter genes containing continuous HSEs. HSF2, whose function is implicated in neuronal specification and spermatogenesis, exhibited a slightly higher binding affinity to discontinuous HSEs than did HSF1. HSF4, a protein required for ocular lens development, efficiently recognized discontinuous HSEs in a trimerization-dependent manner. Among four human gamma-crystallin genes encoding structural proteins of the lens, heat-induced HSF1 preferred HSEs on the gammaA-crystallin and gammaB-crystallin promoters, whereas HSF4 preferred HSE on the gammaC-crystallin promoter. These results suggest that the HSE architecture is an important determinant of which HSF members regulate genes in diverse cellular processes.

Full Text Available E2F transcription factors and their regulatory partners, the pocket proteins (PPs, have emerged as essential regulators of stem cell fate control in a number of lineages. In mammals, this role extends from both pluripotent stem cells to those encompassing all embryonic germ layers, as well as extra-embryonic lineages. E2F/PP-mediated regulation of stem cell decisions is highly evolutionarilyconserved, and is likely a pivotal biological mechanism underlying stem cell homeostasis. This has immense implications for organismal development, tissue maintenance and regeneration. In this article, we discuss the roles of E2F factors and PPs in stem cell populations, focusing on mammalian systems. We discuss emerging findings that position the E2F and PP families as widespread and dynamic epigenetic regulators of cell fate decisions. Additionally, we focus on the ever expanding landscape of E2F/PP target genes, and explore the possibility that E2Fs are not simply regulators of general ‘multi-purpose’ cell fate genes but can execute tissue- and cell type-specific gene regulatory programs.

The TEAD (transcriptional enhancer activator domain) proteins share an evolutionarilyconserved DNA-binding TEA domain, which binds to the MCAT cis-acting regulatory element. Previous studies have shown that TEAD proteins are involved in regulating the expression of smooth muscle α-actin. However, it remains undetermined whether TEAD proteins play a broader role in regulating expression of other genes in vascular smooth muscle cells. In this study, we show that the expression of TEAD1 is significantly induced during smooth muscle cell phenotypic modulation and negatively correlates with smooth muscle-specific gene expression. We further demonstrate that TEAD1 plays a novel role in suppressing expression of smooth muscle-specific genes, including smooth muscle α-actin, by abolishing the promyogenic function of myocardin, a key mediator of smooth muscle differentiation. Mechanistically, we found that TEAD1 competes with myocardin for binding to serum response factor (SRF), resulting in disruption of myocardin and SRF interactions and thereby attenuating expression of smooth muscle-specific genes. This study provides the first evidence demonstrating that TEAD1 is a novel general repressor of smooth muscle-specific gene expression through interfering with myocardin binding to SRF.

members strengthen the monitoring practices to their advantage, and to some extent move them beyond the reach of government agencies and conservation and development practitioners. This has led to outcomes that are of greater social and strategic value to communities than the original 'planned' benefits......, although the monitoring scheme has also to some extent become dominated by local 'conservation elites' who negotiate the terrain between the state and other community members. Our findings suggest that we need to move beyond simplistic assumptions of community strategies and incentives in participatory...... conservation and allow for more adaptive and politically explicit governance spaces in protected area management....

There is considerable evidence that transcription does not occur homogeneously or diffusely throughout the nucleus, but rather at a number of specialized, discrete sites termed transcription factories. The factories are composed of ~4–30 RNA polymerase molecules, and are associated with many other molecules involved in transcriptional activation and mRNA processing. Some data suggest that the polymerase molecules within a factory remain stationary relative to the transcribed DNA, which is thought to be reeled through the factory site. There is also some evidence that transcription factories could help organize chromatin and nuclear structure, contributing to both the formation of chromatin loops and the clustering of active and co-regulated genes. PMID:23109938

Full Text Available In plants and animals, nucleotide-binding domain and leucine-rich repeats (NLR–containing proteins play pivotal roles in innate immunity. Despite their similar biological functions and protein architecture, comparative genome-wide analyses of NLRs and genes encoding NLR-like proteins suggest that plant and animal NLRs have independently arisen in evolution. Furthermore, the demonstration of interfamily transfer of plant NLR functions from their original species to phylogenetically distant species implies evolutionary conservation of the underlying immune principle across plant taxonomy. In this review we discuss plant NLR evolution and summarise recent insights into plant NLR signalling mechanisms, which might constitute evolutionarilyconserved NLR-mediated immune mechanisms.

In this paper we consider how conservation has arisen as a key aspect of the reaction to human-initiated degradation and disappearance of ecosystems, wild lands. and wildlife. Concern over species extinction is given an historical perspective which shows the way in which pressure on wild and natural aspects of global ecology have changed in recent centuries. The role of conservation in the struggle to protect the environment is then analysed using underlying ethical arguments behind the econo...

Full Text Available Although a large proportion of human transcription occurs outside the boundaries of known genes, the functional significance of this transcription remains unknown. We have compared the expression patterns of known genes as well as intergenic transcripts within the ENCODE regions between humans and chimpanzees in brain, heart, testis, and lymphoblastoid cell lines. We find that intergenic transcripts show patterns of tissue-specific conservation of their expression, which are comparable to exonic transcripts of known genes. This suggests that intergenic transcripts are subject to functional constraints that restrict their rate of evolutionary change as well as putative positive selection to an extent comparable to that of classical protein-coding genes. In brain and testis, we find that part of this intergenic transcription is caused by widespread use of alternative promoters. Further, we find that about half of the expression differences between humans and chimpanzees are due to intergenic transcripts.

Although a large proportion of human transcription occurs outside the boundaries of known genes, the functional significance of this transcription remains unknown. We have compared the expression patterns of known genes as well as intergenic transcripts within the ENCODE regions between humans and chimpanzees in brain, heart, testis, and lymphoblastoid cell lines. We find that intergenic transcripts show patterns of tissue-specific conservation of their expression, which are comparable to exonic transcripts of known genes. This suggests that intergenic transcripts are subject to functional constraints that restrict their rate of evolutionary change as well as putative positive selection to an extent comparable to that of classical protein-coding genes. In brain and testis, we find that part of this intergenic transcription is caused by widespread use of alternative promoters. Further, we find that about half of the expression differences between humans and chimpanzees are due to intergenic transcripts. PMID:17040132

Marsupials and monotremes represent evolutionarily divergent lineages from the majority of extant mammals which are eutherian, or placental, mammals. Monotremes possess multiple X and Y chromosomes that appear to have arisen independently of eutherian and marsupial sex chromosomes. Dosage compensation of X-linked genes occurs in monotremes on a gene-by-gene basis, rather than through chromosome-wide silencing, as is the case in eutherians and marsupials. Specifically, studies in the platypus have shown that for any given X-linked gene, a specific proportion of nuclei within a cell population will silence one locus, with the percentage of cells undergoing inactivation at that locus being highly gene-specific. Hence, it is perhaps not surprising that the expression level of X-linked genes in female platypus is almost double that in males. This is in contrast to the situation in marsupials where one of the two X chromosomes is inactivated in females by the long non-coding RNA RSX, a functional analogue of the eutherian XIST. However, marsupial X chromosome inactivation differs from that seen in eutherians in that it is exclusively the paternal X chromosome that is silenced. In addition, marsupials appear to have globally upregulated X-linked gene expression in both sexes, thus balancing their expression levels with those of the autosomes, a process initially proposed by Ohno in 1967 as being a fundamental component of the X chromosome dosage compensation mechanism but which may not have evolved in eutherians.

Full Text Available Abstract Background Corals are notoriously difficult to identify at the species-level due to few diagnostic characters and variable skeletal morphology. This 'coral species problem' is an impediment to understanding the evolution and biodiversity of this important and threatened group of organisms. We examined the evolution of the nuclear ribosomal internal transcribed spacer (ITS and mitochondrial markers (COI, putative control region in Porites, one of the most taxonomically challenging and ecologically important genera of reef-building corals. Results Nuclear and mitochondrial markers were congruent, clearly resolving many traditionally recognized species; however, branching and mounding varieties were genetically indistinguishable within at least two clades, and specimens matching the description of 'Porites lutea' sorted into three genetically divergent groups. Corallite-level features were generally concordant with genetic groups, although hyper-variability in one group (Clade I overlapped and obscured several others, and Synarea (previously thought to be a separate subgenus was closely related to congeners despite its unique morphology. Scanning electron microscopy revealed subtle differences between genetic groups that may have been overlooked previously as taxonomic characters. Conclusion This study demonstrates that the coral skeleton can be remarkably evolutionarily plastic, which may explain some taxonomic difficulties, and obscure underlying patterns of endemism and diversity.

Background Corals are notoriously difficult to identify at the species-level due to few diagnostic characters and variable skeletal morphology. This 'coral species problem' is an impediment to understanding the evolution and biodiversity of this important and threatened group of organisms. We examined the evolution of the nuclear ribosomal internal transcribed spacer (ITS) and mitochondrial markers (COI, putative control region) in Porites, one of the most taxonomically challenging and ecologically important genera of reef-building corals. Results Nuclear and mitochondrial markers were congruent, clearly resolving many traditionally recognized species; however, branching and mounding varieties were genetically indistinguishable within at least two clades, and specimens matching the description of 'Porites lutea' sorted into three genetically divergent groups. Corallite-level features were generally concordant with genetic groups, although hyper-variability in one group (Clade I) overlapped and obscured several others, and Synarea (previously thought to be a separate subgenus) was closely related to congeners despite its unique morphology. Scanning electron microscopy revealed subtle differences between genetic groups that may have been overlooked previously as taxonomic characters. Conclusion This study demonstrates that the coral skeleton can be remarkably evolutionarily plastic, which may explain some taxonomic difficulties, and obscure underlying patterns of endemism and diversity. PMID:19239678

Full Text Available Abstract The problem of functional annotation based on homology modeling is primary to current bioinformatics research. Researchers have noted regularities in sequence, structure and even chromosome organization that allow valid functional cross-annotation. However, these methods provide a lot of false negatives due to limited specificity inherent in the system. We want to create an evolutionarily inspired organization of data that would approach the issue of structure-function correlation from a new, probabilistic perspective. Such organization has possible applications in phylogeny, modeling of functional evolution and structural determination. ELISA (Evolutionary Lineage Inferred from Structural Analysis, http://romi.bu.edu/elisa is an online database that combines functional annotation with structure and sequence homology modeling to place proteins into sequence-structure-function "neighborhoods". The atomic unit of the database is a set of sequences and structural templates that those sequences encode. A graph that is built from the structural comparison of these templates is called PDUG (protein domain universe graph. We introduce a method of functional inference through a probabilistic calculation done on an arbitrary set of PDUG nodes. Further, all PDUG structures are mapped onto all fully sequenced proteomes allowing an easy interface for evolutionary analysis and research into comparative proteomics. ELISA is the first database with applicability to evolutionary structural genomics explicitly in mind. Availability: The database is available at http://romi.bu.edu/elisa.

A key assumption of the ideal free distribution (IFD) is that there are no costs in moving between habitat patches. However, because many populations exhibit more or less continuous population movement between patches and traveling cost is a frequent factor, it is important to determine the effects of costs on expected population movement patterns and spatial distributions. We consider a food chain (tritrophic or bitrophic) in which one species moves between patches, with energy cost or mortality risk in movement. In the two-patch case, assuming forced movement in one direction, an evolutionarily stable strategy requires bidirectional movement, even if costs during movement are high. In the N-patch case, assuming that at least one patch is linked bidirectionally to all other patches, optimal movement rates can lead to source-sink dynamics where patches with negative growth rates are maintained by other patches with positive growth rates. As well, dispersal between patches is not balanced (even in the two-patch case), leading to a deviation from the IFD. Our results indicate that cost-associated forced movement can have important consequences for spatial metapopulation dynamics. Relevance to marine reserve design and the study of stream communities subject to drift is discussed.

A key assumption of the ideal free distribution (IFD) is that there are no costs in moving between habitat patches. However, because many populations exhibit more or less continuous population movement between patches and traveling cost is a frequent factor, it is important to determine the effects of costs on expected population movement patterns and spatial distributions. We consider a food chain (tritrophic or bitrophic) in which one species moves between patches, with energy cost or mortality risk in movement. In the two-patch case, assuming forced movement in one direction, an evolutionarily stable strategy requires bidirectional movement, even if costs during movement are high. In the N-patch case, assuming that at least one patch is linked bidirectionally to all other patches, optimal movement rates can lead to source-sink dynamics where patches with negative growth rates are maintained by other patches with positive growth rates. As well, dispersal between patches is not balanced (even in the two-patch case), leading to a deviation from the IFD. Our results indicate that cost-associated forced movement can have important consequences for spatial metapopulation dynamics. Relevance to marine reserve design and the study of stream communities subject to drift is discussed. ?? 2011 by The University of Chicago.

Endocrinologists can make significant contributions to conservation biology by helping to understand the mechanisms by which organisms cope with changing environments. Field endocrine techniques have advanced rapidly in recent years and can provide substantial information on the growth, stress, and reproductive status of individual animals, thereby providing insight into current and future responses of populations to changes in the environment. Environmental stressors and reproductive status can be detected nonlethally by measuring a number of endocrine-related endpoints, including steroids in plasma, living and nonliving tissue, urine, and feces. Information on the environmental or endocrine requirements of individual species for normal growth, development, and reproduction will provide critical information for species and ecosystem conservation. For many taxa, basic information on endocrinology is lacking, and advances in conservation endocrinology will require approaches that are both “basic” and “applied” and include integration of laboratory and field approaches.

The structure of complex transcriptional regulatory networks has been studied extensively in certain model organisms. However, the evolutionary dynamics of these networks across organisms, which would reveal important principles of adaptive regulatory changes, are poorly understood. We use the known transcriptional regulatory network of Escherichia coli to analyse the conservation patterns of this network across 175 prokaryotic genomes, and predict components of the regulatory networks for these organisms. We observe that transcription factors are typically less conserved than their target genes and evolve independently of them, with different organisms evolving distinct repertoires of transcription factors responding to specific signals. We show that prokaryotic transcriptional regulatory networks have evolved principally through widespread tinkering of transcriptional interactions at the local level by embedding orthologous genes in different types of regulatory motifs. Different transcription factors have emerged independently as dominant regulatory hubs in various organisms, suggesting that they have convergently acquired similar network structures approximating a scale-free topology. We note that organisms with similar lifestyles across a wide phylogenetic range tend to conserve equivalent interactions and network motifs. Thus, organism-specific optimal network designs appear to have evolved due to selection for specific transcription factors and transcriptional interactions, allowing responses to prevalent environmental stimuli. The methods for biological network analysis introduced here can be applied generally to study other networks, and these predictions can be used to guide specific experiments.

The four Toll/IL-1R domain-containing adaptor proteins MyD88, MAL, TRIF, and TRAM are well established as essential mediators of TLR signaling and gene induction following microbial detection. In contrast, the function of the fifth, most evolutionarilyconserved Toll/IL-1R adaptor, sterile α and HEAT/Armadillo motif-containing protein (SARM), has remained more elusive. Recent studies of Sarm(-/-) mice have highlighted a role for SARM in stress-induced neuronal cell death and immune responses in the CNS. However, whether SARM has a role in immune responses in peripheral myeloid immune cells is less clear. Thus, we characterized TLR-induced cytokine responses in SARM-deficient murine macrophages and discovered a requirement for SARM in CCL5 production, whereas gene induction of TNF, IL-1β, CCL2, and CXCL10 were SARM-independent. SARM was not required for TLR-induced activation of MAPKs or of transcription factors implicated in CCL5 induction, namely NF-κB and IFN regulatory factors, nor for Ccl5 mRNA stability or splicing. However, SARM was critical for the recruitment of transcription factors and of RNA polymerase II to the Ccl5 promoter. Strikingly, the requirement of SARM for CCL5 induction was not restricted to TLR pathways, as it was also apparent in cytosolic RNA and DNA responses. Thus, this study identifies a new role for SARM in CCL5 expression in macrophages.

Full Text Available The terpene trilactones (TTLs are believed to be important for the pharmacological properties of Ginkgo biloba leaves extract. 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR is a critical enzyme involved in the biosynthetic pathway of TTLs. In this study, an 1.2-kb fragment of 5’ flanking region of the HMGR gene (GbHMGR, was isolated from G. biloba by genome walking. Extensive sequence analysis revealed the presence of evolutionarilyconserved and over-represented putative cis-acting elements in light-regulated transcription, hormone signaling (gibberellic acid, jasmonate and salicylic acid, elicitor and stress responses (cold/dehydration responses, and plant defense signaling (W-box/WRKY that are common to the promoter region of GbHMGR. EMSA analysis suggested possible functionality of W-box in GbHMGR promoter region. The behavior of gene transcripts in ginkgo callus upon light, low temperature, MeJA and SA treatments further verified the regulatory function of GbHMGR promoter. A significant positive relationship between gene expression level and total TTL contents suggested that GbHMGR might be one of key genes involved in TTL biosynthesis in G. biloba.

Identifying the genomic regions bound by sequence-specific regulatory factors is central both to deciphering the complex DNA cis-regulatory code that controls transcription in metazoans and to determining the range of genes that shape animal morphogenesis. Here, we use whole-genome tiling arrays to map sequences bound in Drosophila melanogaster embryos by the six maternal and gap transcription factors that initiate anterior-posterior patterning. We find that these sequence-specific DNA binding proteins bind with quantitatively different specificities to highly overlapping sets of several thousand genomic regions in blastoderm embryos. Specific high- and moderate-affinity in vitro recognition sequences for each factor are enriched in bound regions. This enrichment, however, is not sufficient to explain the pattern of binding in vivo and varies in a context-dependent manner, demonstrating that higher-order rules must govern targeting of transcription factors. The more highly bound regions include all of the over forty well-characterized enhancers known to respond to these factors as well as several hundred putative new cis-regulatory modules clustered near developmental regulators and other genes with patterned expression at this stage of embryogenesis. The new targets include most of the microRNAs (miRNAs) transcribed in the blastoderm, as well as all major zygotically transcribed dorsal-ventral patterning genes, whose expression we show to be quantitatively modulated by anterior-posterior factors. In addition to these highly bound regions, there are several thousand regions that are reproducibly bound at lower levels. However, these poorly bound regions are, collectively, far more distant from genes transcribed in the blastoderm than highly bound regions; are preferentially found in protein-coding sequences; and are less conserved than highly bound regions. Together these observations suggest that many of these poorly-bound regions are not involved in early

Full Text Available Identifying the genomic regions bound by sequence-specific regulatory factors is central both to deciphering the complex DNA cis-regulatory code that controls transcription in metazoans and to determining the range of genes that shape animal morphogenesis. We used whole-genome tiling arrays to map sequences bound in Drosophila melanogaster embryos by the six maternal and gap transcription factors that initiate anterior-posterior patterning. We find that these sequence-specific DNA binding proteins bind with quantitatively different specificities to highly overlapping sets of several thousand genomic regions in blastoderm embryos. Specific high- and moderate-affinity in vitro recognition sequences for each factor are enriched in bound regions. This enrichment, however, is not sufficient to explain the pattern of binding in vivo and varies in a context-dependent manner, demonstrating that higher-order rules must govern targeting of transcription factors. The more highly bound regions include all of the over 40 well-characterized enhancers known to respond to these factors as well as several hundred putative new cis-regulatory modules clustered near developmental regulators and other genes with patterned expression at this stage of embryogenesis. The new targets include most of the microRNAs (miRNAs transcribed in the blastoderm, as well as all major zygotically transcribed dorsal-ventral patterning genes, whose expression we show to be quantitatively modulated by anterior-posterior factors. In addition to these highly bound regions, there are several thousand regions that are reproducibly bound at lower levels. However, these poorly bound regions are, collectively, far more distant from genes transcribed in the blastoderm than highly bound regions; are preferentially found in protein-coding sequences; and are less conserved than highly bound regions. Together these observations suggest that many of these poorly bound regions are not involved in

Full Text Available A universal feature of metazoan sexual development is the generation of oocyte P granules that withhold certain mRNA species from translation to provide coding potential for proteins during early post-fertilization development. Stabilisation of translationally quiescent mRNA pools in female Plasmodium gametocytes depends on the RNA helicase DOZI, but the molecular machinery involved in the silencing of transcripts in these protozoans is unknown. Using affinity purification coupled with mass-spectrometric analysis we identify a messenger ribonucleoprotein (mRNP from Plasmodium berghei gametocytes defined by DOZI and the Sm-like factor CITH (homolog of worm CAR-I and fly Trailer Hitch. This mRNP includes 16 major factors, including proteins with homologies to components of metazoan P granules and archaeal proteins. Containing translationally silent transcripts, this mRNP integrates eIF4E and poly(A-binding protein but excludes P body RNA degradation factors and translation-initiation promoting eIF4G. Gene deletion mutants of 2 core components of this mRNP (DOZI and CITH are fertilization-competent, but zygotes fail to develop into ookinetes in a female gametocyte-mutant fashion. Through RNA-immunoprecipitation and global expression profiling of CITH-KO mutants we highlight CITH as a crucial repressor of maternally supplied mRNAs. Our data define Plasmodium P granules as an ancient mRNP whose protein core has remained evolutionarilyconserved from single-cell organisms to germ cells of multi-cellular animals and stores translationally silent mRNAs that are critical for early post-fertilization development during the initial stages of mosquito infection. Therefore, translational repression may offer avenues as a target for the generation of transmission blocking strategies and contribute to limiting the spread of malaria.

Some people only think about conservation on Earth Day. Being in the "art business" however, this author is always conscious of the many products she thinks get wasted when they could be reused, recycled, and restored--especially in a school building and art room. In this article, she describes an art lesson that allows students to paint…

Each of the six instructional units deals with one aspect of conservation: forests, water, rangeland, minerals (petroleum), and soil. The area of the elementary school curriculum with which each correlates is indicated. Lists of general and specific objectives are followed by suggested teaching procedures, including ideas for introducing the…

Instructional units deal with each aspect of conservation: forests, wildlife, rangelands, water, minerals, and soil. The area of the secondary school curriculum with which each is correlated is indicated. Lists of general and specific objectives are followed by suggested teaching procedures, including ideas for introducing the topic, questions to…

Recent studies have demonstrated extensive transcriptional activity across the human genome, a substantial fraction of which is not associated with any functional annotation. However, very little is known regarding the post-transcriptional processes that operate within the different classes of RNA molecules. To characterize the post-transcriptional properties of expressed sequences from human chromosome 21 (HSA21), we separated RNA molecules from three cell lines (GM06990, HeLa S3, and SK-N-AS) according to their ribosome content by sucrose gradient fractionation. Polyribosomal-associated RNA and total RNA were subsequently hybridized to genomic tiling arrays. We found that approximately 50% of the transcriptional signals were located outside of annotated exons and were considered as TARs (transcriptionally active regions). Although TARs were observed among polysome-associated RNAs, RT-PCR and RACE experiments revealed that approximately 40% were likely to represent nonspecific cross-hybridization artifacts. Bioinformatics discrimination of TARs according to conservation and sequence complexity allowed us to identify a set of high-confidence TARs. This set of TARs was significantly depleted in the polysomes, suggesting that it was not likely to be involved in translation. Analysis of polysome representation of RefSeq exons showed that at least 15% of RefSeq transcripts undergo significant post-transcriptional regulation in at least two of the three cell lines tested. Among the regulated transcripts, enrichment analysis revealed an over-representation of genes involved in Alzheimer's disease (AD), including APP and the BACE1 protease that cleaves APP to produce the pathogenic beta 42 peptide. We demonstrate that the combination of RNA fractionation and tiling arrays is a powerful method to assess the transcriptional and post-transcriptional properties of genomic regions.

Full Text Available Abstract Background Structural genomics projects such as the Protein Structure Initiative (PSI yield many new structures, but often these have no known molecular functions. One approach to recover this information is to use 3D templates – structure-function motifs that consist of a few functionally critical amino acids and may suggest functional similarity when geometrically matched to other structures. Since experimentally determined functional sites are not common enough to define 3D templates on a large scale, this work tests a computational strategy to select relevant residues for 3D templates. Results Based on evolutionary information and heuristics, an Evolutionary Trace Annotation (ETA pipeline built templates for 98 enzymes, half taken from the PSI, and sought matches in a non-redundant structure database. On average each template matched 2.7 distinct proteins, of which 2.0 share the first three Enzyme Commission digits as the template's enzyme of origin. In many cases (61% a single most likely function could be predicted as the annotation with the most matches, and in these cases such a plurality vote identified the correct function with 87% accuracy. ETA was also found to be complementary to sequence homology-based annotations. When matches are required to both geometrically match the 3D template and to be sequence homologs found by BLAST or PSI-BLAST, the annotation accuracy is greater than either method alone, especially in the region of lower sequence identity where homology-based annotations are least reliable. Conclusion These data suggest that knowledge of evolutionarily important residues improves functional annotation among distant enzyme homologs. Since, unlike other 3D template approaches, the ETA method bypasses the need for experimental knowledge of the catalytic mechanism, it should prove a useful, large scale, and general adjunct to combine with other methods to decipher protein function in the structural proteome.

Piriformospora indica, an endophytic fungus of the Sebacinaceae family, promotes growth of Arabidopsis and tobacco seedlings and stimulates nitrogen accumulation and the expression of the genes for nitrate reductase and the starch-degrading enzyme glucan-water dikinase (SEX1) in roots. Neither growth promotion nor stimulation of the two enzymes requires heterotrimeric G proteins. P. indica also stimulates the expression of the uidA gene under the control of the Arabidopsis nitrate reductase (Nia2) promoter in transgenic tobacco seedlings. At least two regions (-470/-439 and -103/-89) are important for Nia2 promoter activity in tobacco roots. One of the regions contains an element, ATGATAGATAAT, that binds to a homeodomain transcription factor in vitro. The message for this transcription factor is up-regulated by P. indica. The transcription factor also binds to a CTGATAGATCT segment in the SEX1 promoter in vitro. We propose that the growth-promoting effect initiated by P. indica is accompanied by a co-regulated stimulation of enzymes involved in nitrate and starch metabolisms.

Regulation of transcription elongation via pausing of RNA polymerase has multiple physiological roles. The pausing mechanism depends on the sequence heterogeneity of the DNA being transcribed, as well as on certain interactions of polymerase with specific DNA sequences. In order to describe the mechanism of regulation, we introduce the concept of heterogeneity into the previously proposed alternative models of elongation, power stroke and Brownian ratchet. We also discuss molecular origins and physiological significances of the heterogeneity. PMID:25764114

Herons are large, popular and, in many cases, spectacular birds found in wetlands world-wide, both tropical and temperate, natural and man-made. Some populations are very small and localized, some have decreased, some have expanded their ranges, and a few are pests of human activities. In the fifteen years since the publication of the latest monographic treatment of the family, The Herons Handbook, there has been a tremendous increase in our knowledge of heron status and conservation requirements, set against a backdrop of increasing concern about the future of the world?s wetland habitats. This book provides a comprehensive update following two distinct threads. The status and conservation needs of herons are first presented on a regional basis, in a series of chapters set at a continental or subcontinental scale. Over 200 biologists and heron conservationists have contributed to the data summarized here, and the very latest census and survey results provide the most up-to-date and detailed picture of heron populations currently available. Chapters discussing several critical issues in heron conservation follow, tending to focus on the international nature of the problems.

The Snail family of transcription factors are core inducers of epithelial-to-mesenchymal transition (EMT). Here we show that the F-box protein FBXO11 recognizes and promotes ubiquitin-mediated degradation of multiple Snail family members including Scratch. The association between FBXO11 and Snai1 in vitro is independent of Snai1 phosphorylation. Overexpression of FBXO11 in mesenchymal cells reduces Snail protein abundance and cellular invasiveness. Conversely, depletion of endogenous FBXO11 in epithelial cancer cells causes Snail protein accumulation, EMT, and tumor invasion, as well as loss of estrogen receptor expression in breast cancer cells. Expression of FBXO11 is downregulated by EMT-inducing signals TGFβ and nickel. In human cancer, high FBXO11 levels correlate with expression of epithelial markers and favorable prognosis. The results suggest that FBXO11 sustains the epithelial state and inhibits cancer progression. Inactivation of FBXO11 in mice leads to neonatal lethality, epidermal thickening, and increased Snail protein levels in epidermis, validating that FBXO11 is a physiological ubiquitin ligase of Snail. Moreover, in C. elegans, the FBXO11 mutant phenotype is attributed to the Snail factors as it is suppressed by inactivation/depletion of Snail homologs. Collectively, these findings suggest that the FBXO11-Snail regulatory axis is evolutionarilyconserved and critically governs carcinoma progression and mammalian epidermal development. PMID:25827072

Yeast prions are non-Mendelian genetic elements that are conferred by altered and self-propagating protein conformations. Such a protein conformation-based transmission is similar to that of PrP(Sc), the infectious protein responsible for prion diseases. Despite recent progress in understanding the molecular nature and epigenetic transmission of prions, the underlying mechanisms governing prion conformational switch and determining prion "strains" are not understood. We report here that the evolutionarilyconserved heat-shock transcription factor (HSF) strongly influences yeast prion formation and strain determination. An hsf1 mutant lacking the amino-terminal activation domain inhibits the yeast prion [PSI+] formation whereas a mutant lacking the carboxyl-terminal activation domain promotes [PSI+] formation. Moreover, specific [PSI+] strains are preferentially formed in these mutants, demonstrating the importance of genetic makeup in determining de novo appearance of prion strains. Although these hsf1 mutants preferentially support the formation of certain [PSI+] strains, they are capable of receiving and faithfully propagating nonpreferable strains, suggesting that prion initiation and propagation are distinct processes requiring different cellular components. Our findings establish the importance of HSF in prion initiation and strain determination and imply a similar regulatory role of mammalian HSFs in the complex etiology of prion disease.

The sequence of the 8,600-base-pair HindIII H fragment, located at the center of the vaccinia virus genome, was determined to analyze several late genes. Seven major complete open reading frames (ORFs) and two that started from or continued into adjacent DNA segments were identified. ORFs were closely spaced and present on both DNA strands. Some adjacent ORFs had oppositely oriented overlapping termination codons or contiguous stop and start codons. Nucleotide compositional analysis indicated that the A-T frequency was consistently lowest in the first codon position. The sizes of the polypeptides predicted from the DNA sequence were compared with those determined by polyacrylamide gel electrophoresis of cell-free translation products of mRNAs selected by hybridization to cloned single-stranded DNA segments or synthesized in vitro by bacteriophage T7 RNA polymerase. Six transcripts that initiated within the HindIII H DNA fragment were detected, and of these, four were synthesized only at late times, one was synthesized only early, and one was synthesized early and late. The sites on the genome corresponding to the 5' ends of the transcripts were located by high-resolution nuclease S1 analysis. For late genes, the transcriptional and translational initiation sites mapped within a few nucleotides of each other, and in each case the sequence TAAATGG occurred at the start of the ORF. The extremely short leader and the absence of A or G in the -3 position, relative to the first nucleotide of the initiation codon, distinguishes the majority of vaccinia virus late genes from eucaryotic and vaccinia virus early genes.

Cytoplasmic polyadenylylation is an evolutionarilyconserved mechanism involved in the translational activation of a set of maternal messenger RNAs (mRNAs) during early development. In this report, we show by interspecies injections that Xenopus and mouse use the same regulatory sequences to control cytoplasmic poly(A) addition during meiotic maturation. Similarly, Xenopus and Drosophila embryos exploit functionally conserved signals to regulate polyadenylylation during early post-fertilization development. These experiments demonstrate that the sequence elements that govern cytoplasmic polyadenylylation, and hence one form of translational activation, function across species. We infer that the requisite regulatory sequence elements, and likely the trans-acting components with which they interact, have been conserved since the divergence of vertebrates and arthropods.

Improper gene regulation is implicated in reproductive isolation, but its genetic and molecular bases are unknown. We previously reported that a mouse inter-subspecific X chromosome substitution strain shows reproductive isolation characterized by male-specific sterility due to disruption of meiotic entry in spermatogenesis. Here, we conducted comprehensive transcriptional profiling of the testicular cells of this strain by microarray. The results clearly revealed gross misregulation of gene expression in the substituted donor X chromosome. Such misregulation occurred prior to detectable spermatogenetic impairment, suggesting that it is a primal event in reproductive isolation. The misregulation of X-linked genes showed asymmetry; more genes were disproportionally downregulated rather than upregulated. Furthermore, this misregulation subsequently resulted in perturbation of global transcriptional regulation of autosomal genes, probably by cascading deleterious effects. Remarkably, this transcriptional misregulation was substantially restored by introduction of chromosome 1 from the same donor strain as the X chromosome. This finding implies that one of regulatory genes acting in trans for X-linked target genes is located on chromosome 1. This study collectively suggests that regulatory incompatibility is a major cause of reproductive isolation in the X chromosome substitution strain.

Establishment of conservation priorities for primates is a particular concern in the island archipelagos of Southeast Asia, where rates of habitat destruction are among the highest in the world. Conservation programs require knowledge of taxonomic diversity to ensure success. The Philippine tarsier is a flagship species that promotes environmental awareness and a thriving ecotourism economy in the Philippines. However, assessment of its conservation status has been impeded by taxonomic uncertainty, a paucity of field studies, and a lack of vouchered specimens and genetic samples available for study in biodiversity repositories. Consequently, conservation priorities are unclear. In this study we use mitochondrial and nuclear DNA to empirically infer geographic partitioning of genetic variation and to identify evolutionarily distinct lineages for conservation action. The distribution of Philippine tarsier genetic diversity is neither congruent with expectations based on biogeographical patterns documented in other Philippine vertebrates, nor does it agree with the most recent Philippine tarsier taxonomic arrangement. We identify three principal evolutionary lineages that do not correspond to the currently recognized subspecies, highlight the discovery of a novel cryptic and range-restricted subcenter of genetic variation in an unanticipated part of the archipelago, and identify additional geographically structured genetic variation that should be the focus of future studies and conservation action. Conservation of this flagship species necessitates establishment of protected areas and targeted conservation programs within the range of each genetically distinct variant of the Philippine tarsier.

Transcription of eukaryotic protein-coding genes commences with the assembly of a conserved initiation complex, which consists of RNA polymerase II (Pol II) and the general transcription factors, at promoter DNA. After two decades of research, the structural basis of transcription initiation is emerging. Crystal structures of many components of the initiation complex have been resolved, and structural information on Pol II complexes with general transcription factors has recently been obtaine...

Full Text Available Abstract Background Sin3A is an evolutionarilyconservedtranscriptional repressor which regulates gene expression as part of the multi-protein Sin3 repressive complex. It functions as a scaffold upon which proteins with enzymatic activity dock, including chromatin modifying histone deacetylases. Although regulation of transcription by Sin3A has been studied in detail, little is understood about the function of Sin3A in cancer cells. We previously showed that Sin3A is expressed in breast cancer cells and is a repressor of estrogen receptor-alpha (ERα, ESR1 gene expression. Here, we expand our previous studies to elucidate the function of Sin3A in the control of gene expression and growth of breast cancer cells. Results Analysis of gene expression following knockdown of Sin3A revealed changes in both basal and regulated gene transcription. Genes of known importance in breast cancer and estrogen signaling, including ERBB2, PGR, MYC, CLU, and NCOA2, were among those identified as Sin3A-responsive. The mechanism of Sin3A action varied among genes and was found to be mediated through both HDAC1/2 -dependent and -independent activities. Loss of Sin3A inhibited breast cancer cell growth by increasing apoptosis without affecting cell cycle progression. Analysis of both ERα-positive and ERα-negative cell lines revealed that the effects of Sin3A on growth were cell-type specific, as Sin3A expression promoted maximum growth of only the ERα-positive cells, and, notably, Sin3A protein itself was increased by estrogen. Further gene expression experiments revealed that Sin3A repressed expression of key apoptotic genes, including TRAIL, TRAILR1, CASP10, and APAF1, in ERα-positive, but not ERα-negative, cell lines, which could provide a mechanistic explanation for cell-type differences in growth. Conclusions This study identifies Sin3A as a regulator of gene expression, survival, and growth in ERα-positive breast cancer cells. Sin3A regulates the

The heat shock sigma factor (sigma(32) in Escherichia coli) directs the bacterial RNA polymerase to promoters of a specific sequence to form a stable complex, competent to initiate transcription of genes whose products mitigate the effects of exposure of the cell to high temperatures. The histidine at position 107 of sigma(32) is at the homologous position of a tryptophan residue at position 433 of the main sigma factor of E. coli, sigma(70). This tryptophan is essential for the strand separation step leading to the formation of the initiation-competent RNA polymerase-promoter complex. The heat shock sigma factors of all gammaproteobacteria sequenced have a histidine at this position, while in the alpha- and deltaproteobacteria, it is a tryptophan. In vitro the alanine-for-histidine substitution at position 107 (H107A) destabilizes complexes between the GroE promoter and RNA polymerase containing sigma(32), implying that H107 plays a role in formation or maintenance of the strand-separated complex. In vivo, the H107A substitution in sigma(32) impedes recovery from heat shock (exposure to 42 degrees C), and it also leads to overexpression at lower temperatures (30 degrees C) of the Flu protein, which is associated with biofilm formation.

Apicomplexan parasites rely on a novel form of actin-based motility called gliding, which depends on parasite actin polymerization, to migrate through their hosts and invade cells. However, parasite actins are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. The molecular basis for parasite actin filament instability and its relationship to gliding motility remain unresolved. We demonstrate that recombinant Toxoplasma (TgACTI) and Plasmodium (PfACTI and PfACTII) actins polymerized into very short filaments in vitro but were induced to form long, stable filaments by addition of equimolar levels of phalloidin. Parasite actins contain a conserved phalloidin-binding site as determined by molecular modeling and computational docking, yet vary in several residues that are predicted to impact filament stability. In particular, two residues were identified that form intermolecular contacts between different protomers in conventional actin filaments and these residues showed non-conservative differences in apicomplexan parasites. Substitution of divergent residues found in TgACTI with those from mammalian actin resulted in formation of longer, more stable filaments in vitro. Expression of these stabilized actins in T. gondii increased sensitivity to the actin-stabilizing compound jasplakinolide and disrupted normal gliding motility in the absence of treatment. These results identify the molecular basis for short, dynamic filaments in apicomplexan parasites and demonstrate that inherent instability of parasite actin filaments is a critical adaptation for gliding motility.

Full Text Available Apicomplexan parasites rely on a novel form of actin-based motility called gliding, which depends on parasite actin polymerization, to migrate through their hosts and invade cells. However, parasite actins are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. The molecular basis for parasite actin filament instability and its relationship to gliding motility remain unresolved. We demonstrate that recombinant Toxoplasma (TgACTI and Plasmodium (PfACTI and PfACTII actins polymerized into very short filaments in vitro but were induced to form long, stable filaments by addition of equimolar levels of phalloidin. Parasite actins contain a conserved phalloidin-binding site as determined by molecular modeling and computational docking, yet vary in several residues that are predicted to impact filament stability. In particular, two residues were identified that form intermolecular contacts between different protomers in conventional actin filaments and these residues showed non-conservative differences in apicomplexan parasites. Substitution of divergent residues found in TgACTI with those from mammalian actin resulted in formation of longer, more stable filaments in vitro. Expression of these stabilized actins in T. gondii increased sensitivity to the actin-stabilizing compound jasplakinolide and disrupted normal gliding motility in the absence of treatment. These results identify the molecular basis for short, dynamic filaments in apicomplexan parasites and demonstrate that inherent instability of parasite actin filaments is a critical adaptation for gliding motility.

Full Text Available BACKGROUND: Ampicillin-resistant Enterococcus faecium (ARE has emerged as a nosocomial pathogen. Here, we quantified ARE carriage in different community sources and determined genetic relatedness with hospital ARE. METHODS AND RESULTS: ARE was recovered from rectal swabs of 24 of 79 (30% dogs, 11 of 85 (13% cats and 0 of 42 horses and from 3 of 40 (8% faecal samples of non-hospitalized humans receiving amoxicillin. Multi-locus Sequence Typing revealed 21 sequence types (STs, including 5 STs frequently associated with hospital-acquired infections. Genes previously found to be enriched in hospital ARE, such as IS16, orf903, orf905, orf907, were highly prevalent in community ARE (≥79%, while genes with a proposed role in pathogenesis, such as esp, hyl and ecbA, were found rarely (≤5% in community isolates. Comparative genome analysis of 2 representative dog isolates revealed that the dog strain of ST192 was evolutionarily closely linked to two previously sequenced hospital ARE, but had, based on gene content, more genes in common with the other, evolutionarily more distantly related, dog strain (ST266. CONCLUSION: ARE were detected in dogs, cats and sporadically in healthy humans, with evolutionary linkage to hospital ARE. Yet, their accessory genome has diversified, probably as a result of niche adaptation.

The central domain of the 200-kDa Lassa virus L protein is a putative RNA-dependent RNA polymerase. N- and C-terminal domains may harbor enzymatic functions important for viral mRNA synthesis, including capping enzymes or cap-snatching endoribonucleases. In the present study, we have employed a large-scale mutagenesis approach to map functionally relevant residues in these regions. The main targets were acidic (Asp and Glu) and basic residues (Lys and Arg) known to form catalytic and binding sites of capping enzymes and endoribonucleases. A total of 149 different mutants were generated and tested in the Lassa virus replicon system. Nearly 25% of evolutionarily highly conserved acidic and basic side chains were dispensable for function of L protein in the replicon context. The vast majority of the remaining mutants had defects in both transcription and replication. Seven residues (Asp-89, Glu-102, Asp-119, Lys-122, Asp-129, Glu-180, and Arg-185) were selectively important for mRNA synthesis. The phenotype was particularly pronounced for Asp-89, Glu-102, and Asp-129, which were indispensable for transcription but could be replaced by a variety of amino acid residues without affecting genome replication. Bioinformatics disclosed the remote similarity of this region to type IIs endonucleases. The mutagenesis was complemented by experiments with the RNA polymerase II inhibitor alpha-amanitin, demonstrating dependence of viral transcription from the cellular mRNA pool. In conclusion, this paper describes an N-terminal region in L protein being important for mRNA, but not genome synthesis. Bioinformatics and cell biological experiments lend support to the hypothesis that this region could be part of a cap-snatching enzyme.

Full Text Available Abstract Background The nuclear receptors (NRs are an important class of transcription factors that are conserved across animal phyla. Canonical NRs consist of a DNA-binding domain (DBD and ligand-binding domain (LBD. While most animals have 20–40 NRs, nematodes of the genus Caenorhabditis have experienced a spectacular proliferation and divergence of NR genes. The LBDs of evolutionarily-conserved Caenorhabditis NRs have diverged sharply from their Drosophila and vertebrate orthologs, while the DBDs have been strongly conserved. The NR2E family of NRs play critical roles in development, especially in the nervous system. In this study, we explore the phylogenetics and function of the NR2E family of Caenorhabditis elegans, using an in vivo assay to test LBD function. Results Phylogenetic analysis reveals that the NR2E family of NRs consists of three broadly-conserved clades of orthologous NRs. In C. elegans, these clades are defined by nhr-67, fax-1 and nhr-239. The vertebrate orthologs of nhr-67 and fax-1 are Tlx and PNR, respectively. While the nhr-239 clade includes orthologs in insects (Hr83, an echinoderm, and a hemichordate, the gene appears to have been lost from vertebrate lineages. The C. elegans and C. briggsae nhr-239 genes have an apparently-truncated and highly-diverged LBD region. An additional C. elegans NR2E gene, nhr-111, appears to be a recently-evolved paralog of fax-1; it is present in C. elegans, but not C. briggsae or other animals with completely-sequenced genomes. Analysis of the relatively unstudied nhr-111 and nhr-239 genes demonstrates that they are both expressed—nhr-111 very broadly and nhr-239 in a small subset of neurons. Analysis of the FAX-1 LBD in an in vivo assay revealed that it is not required for at least some developmental functions. Conclusions Our analysis supports three conserved clades of NR2E receptors, only two of which are represented in vertebrates, indicating three ancestral NR2E genes in the

Full Text Available Abstract Background In eukaryotes, RNA polymerase III (RNAP III transcribes the genes for small RNAs like tRNAs, 5S rRNA, and several viral RNAs, and short interspersed repetitive elements (SINEs. The genes for these RNAs and SINEs have internal promoters that consist of two regions. These two regions are called the A and B blocks. The multisubunit transcription factor TFIIIC is required for transcription initiation of RNAP III; in transcription of tRNAs, the B-block binding subunit of TFIIIC recognizes a promoter. Although internal promoter sequences are conserved in eukaryotes, no evidence of homology between the B-block binding subunits of vertebrates and yeasts has been reported previously. Results Here, I reported the results of PSI-BLAST searches using the B-block binding subunits of human and Shizosacchromyces pombe as queries, showing that the same Arabidopsis proteins were hit with low E-values in both searches. Comparison of the convergent iterative alignments obtained by these PSI-BLAST searches revealed that the vertebrate, yeast, and Arabidopsis proteins have similarities in their N-terminal one-third regions. In these regions, there were three domains with conserved sequence similarities, one located in the N-terminal end region. The N-terminal end region of the B-block binding subunit of Saccharomyces cerevisiae is tentatively identified as a HMG box, which is the DNA binding motif. Although I compared the alignment of the N-terminal end regions of the B-block binding subunits, and their homologs, with that of the HMG boxes, it is not clear whether they are related. Conclusion Molecular phylogenetic analyses using the small subunit rRNA and ubiquitous proteins like actin and α-tubulin, show that fungi are more closely related to animals than either is to plants. Interestingly, the results obtained in this study show that, with respect to the B-block binding subunits of TFIIICs, animals appear to be evolutionarily closer to plants

Periapical cyst is a chronic inflammatory disorder of periradicular tissues. The precise pathological mechanisms involved in periapical cyst enlargement remain unclear. Notch signaling is an evolutionarilyconserved pathway with a regulatory role in cell fate decisions during development and in carcinogenesis. To date, there are no published data available on the expression of Notch signaling components in periapical cysts or any other jaw cyst. In this immunohistochemical study we have examined the expression of the receptor Notch 1, the ligand Delta 1 and the transcription factor HES 1 in the epithelium of well defined periapical cysts. Immunostaining reaction of Notch 1, Delta 1 and HES 1 was observed in the cytoplasm and/or the cytoplasmic membrane and occasionally in the nucleus in the majority of epithelial cells of all periapical cysts. The present observations indicate that Notch pathway is active in the epithelium of periapical cysts. It can be speculated that activation of epithelial cells of periapical cysts is associated with activation of Notch pathway and imply involvement of this pathway in periapical cyst growth and expansion. PMID:21116324

Due to their high degree of conservation, comparisons of DNA sequences among evolutionarily distantly-related genomes permit to identify functional regions in noncoding DNA. Hox genes are optimal candidate sequences for comparative genome analyses, because they are extremely conserved in vertebrates and occur in clusters. We aligned (Pipmaker) the nucleotide sequences of HoxA clusters of tilapia, pufferfish, striped bass, zebrafish, horn shark, human and mouse (over 500 million years of evolutionary distance). We identified several highly conserved intergenic sequences, likely to be important in gene regulation. Only a few of these putative regulatory elements have been previously described as being involved in the regulation of Hox genes, while several others are new elements that might have regulatory functions. The majority of these newly identified putative regulatory elements contain short fragments that are almost completely conserved and are identical to known binding sites for regulatory proteins (Transfac). The conserved intergenic regions located between the most rostrally expressed genes in the developing embryo are longer and better retained through evolution. We document that presumed regulatory sequences are retained differentially in either A or A clusters resulting from a genome duplication in the fish lineage. This observation supports both the hypothesis that the conserved elements are involved in gene regulation and the Duplication-Deletion-Complementation model.

A global strategy is necessary to achieve the level of coordination, synergy and therefore optimization of resources to achieve the broad goal of conserving mammals worldwide. Key elements for the development of such a strategy include: an institutional subject that owns the strategy; broad conservation goals, quantitative targets derived from them and appropriate indicators; data on the distribution of species, their threats, the cost-effectiveness of conservation actions; and a set of methods for the identification of conservation priorities. Previous global mammal research investigated phylogeny, extinction risk, and the species and areas that should be regarded as global conservation priorities. This theme issue presents new key elements: an updated Red List Index, a new list of evolutionarily distinct and globally endangered species, new high-resolution mammal distribution models, a global connectivity analysis and scenarios of future mammal distribution based on climate and land-cover change. Area prioritization schemes account for mammalian phylogeny, governance and cost-benefit of measures to abate habitat loss. Three discussion papers lay the foundations for the development of a global unifying mammal conservation strategy, which should not be further deterred by the knowledge gaps still existing.

A systematic comparative genomic analysis of all archaeal membrane proteins that have been projected to the last archaeal common ancestor gene set led to the identification of several novel components of predicted secretion, membrane remodeling, and protein glycosylation systems. Among other findings, most crenarchaea have been shown to encode highly diverged orthologs of the membrane insertase YidC, which is nearly universal in bacteria, eukaryotes, and euryarchaea. We also identified a vast family of archaeal proteins, including the C-terminal domain of N-glycosylation protein AglD, as membrane flippases homologous to the flippase domain of bacterial multipeptide resistance factor MprF, a bifunctional lysylphosphatidylglycerol synthase and flippase. Additionally, several proteins were predicted to function as membrane transporters. The results of this work, combined with our previous analyses, reveal an unexpected diversity of putative archaeal membrane-associated functional systems that remain to be functionally characterized. A more general conclusion from this work is that the currently available collection of archaeal (and bacterial) genomes could be sufficient to identify (almost) all widespread functional modules and develop experimentally testable predictions of their functions.

Toxoplasma gondii is a protist parasite of warm-blooded animals that causes disease by proliferating intracellularly in muscle and the central nervous system. Previous studies showed that a prolyl 4-hydroxylase related to animal HIFα prolyl hydroxylases is required for optimal parasite proliferation, especially at low O2. We also observed that Pro-154 of Skp1, a subunit of the Skp1/Cullin-1/F-box protein (SCF)-class of E3-ubiquitin ligases, is a natural substrate of this enzyme. In an unrelated protist, Dictyostelium discoideum, Skp1 hydroxyproline is modified by five sugars via the action of three glycosyltransferases, Gnt1, PgtA, and AgtA, which are required for optimal O2-dependent development. We show here that TgSkp1 hydroxyproline is modified by a similar pentasaccharide, based on mass spectrometry, and that assembly of the first three sugars is dependent on Toxoplasma homologs of Gnt1 and PgtA. Reconstitution of the glycosyltransferase reactions in extracts with radioactive sugar nucleotide substrates and appropriate Skp1 glycoforms, followed by chromatographic analysis of acid hydrolysates of the reaction products, confirmed the predicted sugar identities as GlcNAc, Gal, and Fuc. Disruptions of gnt1 or pgtA resulted in decreased parasite growth. Off target effects were excluded based on restoration of the normal glycan chain and growth upon genetic complementation. By analogy to Dictyostelium Skp1, the mechanism may involve regulation of assembly of the SCF complex. Understanding the mechanism of Toxoplasma Skp1 glycosylation is expected to help develop it as a drug target for control of the pathogen, as the glycosyltransferases are absent from mammalian hosts. PMID:26719340

The covalent attachment of ubiquitin to target proteins influences various cellular processes, including DNA repair, NF-kappaB signalling and cell survival. The most common mode of regulation by ubiquitin-conjugation involves specialized ubiquitin-binding proteins that bind to ubiquitylated...... found that the UBA domain is essential for the oncogenic potential of cIAP1, to maintain endothelial cell survival and to protect cells from TNF-alpha-induced apoptosis. Moreover, the UBA domain is required for XIAP and cIAP2-MALT1 to activate NF-kappaB. Our data suggest that the UBA domain of cIAP2......-MALT1 stimulates NF-kappaB signalling by binding to polyubiquitylated NEMO. Significantly, 98% of all cIAP2-MALT1 fusion proteins retain the UBA domain, suggesting that ubiquitin-binding contributes to the oncogenic potential of cIAP2-MALT1 in MALT lymphoma. Our data identify IAPs as ubiquitin...

HIV-1 Vif assembles the Cul5-EloB/C E3 ubiquitin ligase to induce proteasomal degradation of the cellular antiviral APOBEC3 proteins. Detailed structural studies have confirmed critical functional domains in Vif that we have previously identified as important for the interaction of EloB/C, Cul5, and CBFβ. However, the mechanism by which Vif recognizes substrates remains poorly understood. Specific regions of Vif have been identified as being responsible for binding and depleting APOBEC3G and APOBEC3F. Interestingly, we have now identified distinct yet overlapping domains that are required for HIV-1 Vif-mediated G2/M-phase cell cycle arrest and APOBEC3H degradation, but not for the inactivation of APOBEC3G or APOBEC3F. Surprisingly, Vif molecules from primary HIV-1 variants that caused G2/M arrest were unable to inactivate APOBEC3H; on the other hand, HIV-1 Vif variants that could inactivate APOBEC3H were unable to induce G2/M arrest. All of these Vif variants still maintained the ability to inactivate APOBEC3G/F. Thus, primary HIV-1 variants have evolved to possess distinct functional activities that allow them to suppress APOBEC3H or cause G2 cell cycle arrest, using mutually exclusive interface domains. APOBEC3H depletion and G2 arrest are apparently evolutionary selected features that cannot co-exist on a single Vif molecule. The existence and persistence of both types of HIV-1 Vif variant suggests the importance of APOBEC3H suppression and cell cycle regulation for HIV-1's survival in vivo.

Toxoplasma gondii is a protist parasite of warm-blooded animals that causes disease by proliferating intracellularly in muscle and the central nervous system. Previous studies showed that a prolyl 4-hydroxylase related to animal HIFα prolyl hydroxylases is required for optimal parasite proliferation, especially at low O2. We also observed that Pro-154 of Skp1, a subunit of the Skp1/Cullin-1/F-box protein (SCF)-class of E3-ubiquitin ligases, is a natural substrate of this enzyme. In an unrelated protist, Dictyostelium discoideum, Skp1 hydroxyproline is modified by five sugars via the action of three glycosyltransferases, Gnt1, PgtA, and AgtA, which are required for optimal O2-dependent development. We show here that TgSkp1 hydroxyproline is modified by a similar pentasaccharide, based on mass spectrometry, and that assembly of the first three sugars is dependent on Toxoplasma homologs of Gnt1 and PgtA. Reconstitution of the glycosyltransferase reactions in extracts with radioactive sugar nucleotide substrates and appropriate Skp1 glycoforms, followed by chromatographic analysis of acid hydrolysates of the reaction products, confirmed the predicted sugar identities as GlcNAc, Gal, and Fuc. Disruptions of gnt1 or pgtA resulted in decreased parasite growth. Off target effects were excluded based on restoration of the normal glycan chain and growth upon genetic complementation. By analogy to Dictyostelium Skp1, the mechanism may involve regulation of assembly of the SCF complex. Understanding the mechanism of Toxoplasma Skp1 glycosylation is expected to help develop it as a drug target for control of the pathogen, as the glycosyltransferases are absent from mammalian hosts.

from human cells, we obtained two different hTlk1 short hairpin RNAs (shRNAs) and a scrambled control shRNA from the MDACC shRNA core. All shRNAs...identity (EMT)(23). In addition to MCF10A cells, we also treated a Triple Negative Breast tumor cell line, MDA486, with the scrambled control shRNA...24 hours at 20oC. The adults were removed and the number of embryos on the plates counted. The mutant transgenic animals did not appear to be egg

Msx (/msh) family genes encode homeodomain (HD) proteins that control ontogeny in many animal species. We compared the structures of Msx genes from a wide range of Metazoa (Porifera, Cnidaria, Nematoda, Arthropoda, Tardigrada, Platyhelminthes, Mollusca, Brachiopoda, Annelida, Echiura, Echinodermata, Hemichordata, and Chordata) to gain an understanding of the role of these genes in phylogeny. Exon-intron boundary analysis suggested that the position of the intron located N-terminally to the HDs was widely conserved in all the genes examined, including those of cnidarians. Amino acid (aa) sequence comparison revealed 3 new evolutionarilyconserved domains, as well as very strong conservation of the HDs. Two of the three domains were associated with Groucho-like protein binding in both a vertebrate and a cnidarian Msx homolog, suggesting that the interaction between Groucho-like proteins and Msx proteins was established in eumetazoan ancestors. Pairwise comparison among the collected HDs and their C-flanking aa sequences revealed that the degree of sequence conservation varied depending on the animal taxa from which the sequences were derived. Highly conserved Msx genes were identified in the Vertebrata, Cephalochordata, Hemichordata, Echinodermata, Mollusca, Brachiopoda, and Anthozoa. The wide distribution of the conserved sequences in the animal phylogenetic tree suggested that metazoan ancestors had already acquired a set of conserved domains of the current Msx family genes. Interestingly, although strongly conserved sequences were recovered from the Vertebrata, Cephalochordata, and Anthozoa, the sequences from the Urochordata and Hydrozoa showed weak conservation. Because the Vertebrata-Cephalochordata-Urochordata and Anthozoa-Hydrozoa represent sister groups in the Chordata and Cnidaria, respectively, Msx sequence diversification may have occurred differentially in the course of evolution. We speculate that selective loss of the conserved domains in Msx family

Transcription is repressed if a DNA double-strand break (DSB) is introduced in close proximity to a transcriptional activation site at least in part by H2A-ubiquitination. While ATM signaling is involved, how it controls H2A-ubiquitination remains unclear. Here, we identify that, in response to DSBs, a transcriptional elongation factor, ENL (MLLT1), is phosphorylated by ATM at conserved SQ sites. This phosphorylation increases the interaction between ENL and the E3-ubiquitin-ligase complex of Polycomb Repressive Complex 1 (PRC1) via BMI1. This interaction promotes enrichment of PRC1 at transcription elongation sites near DSBs to ubiquitinate H2A leading to transcriptional repression. ENL SQ sites and BMI1 are necessary for KU70 accumulation at DSBs near active transcription sites and cellular resistance to DSBs. Our data suggest that ATM-dependent phosphorylation of ENL functions as switch from elongation to Polycomb-mediated repression to preserve genome integrity.

The NAD(+)-dependent cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12), which is recognized as a key to central carbon metabolism in glycolysis and gluconeogenesis and as an important allozymic polymorphic biomarker, was purified from muscles of two marine species: the skeletal muscle of Sardina pilchardus Walbaum (Teleost, Clupeida) and the incompressible arm muscle of Octopus vulgaris (Mollusca, Cephalopoda). Comparative biochemical studies have revealed that they differ in their subunit molecular masses and in pI values. Partial cDNA sequences corresponding to an internal region of the GapC genes from Sardina and Octopus were obtained by polymerase chain reaction using degenerate primers designed from highly conserved protein motifs. Alignments of the deduced amino acid sequences were used to establish the 3D structures of the active site of two enzymes as well as the phylogenetic relationships of the sardine and octopus enzymes. These two enzymes are the first two GAPDHs characterized so far from teleost fish and cephalopod, respectively. Interestingly, phylogenetic analyses indicated that the sardina GAPDH is in a cluster with the archetypical enzymes from other vertebrates, while the octopus GAPDH comes together with other molluscan sequences in a distant basal assembly closer to bacterial and fungal orthologs, thus suggesting their different evolutionary scenarios.

Full Text Available Transcriptome analysis of polar bear (Ursus maritimus tissues identified sequences with similarity to Porcine Endogenous Retroviruses (PERV. Based on these sequences, four proviral copies and 15 solo long terminal repeats (LTRs of a newly described endogenous retrovirus were characterized from the polar bear draft genome sequence. Closely related sequences were identified by PCR analysis of brown bear (Ursus arctos and black bear (Ursus americanus but were absent in non-Ursinae bear species. The virus was therefore designated UrsusERV. Two distinct groups of LTRs were observed including a recombinant ERV that contained one LTR belonging to each group indicating that genomic invasions by at least two UrsusERV variants have recently occurred. Age estimates based on proviral LTR divergence and conservation of integration sites among ursids suggest the viral group is only a few million years old. The youngest provirus was polar bear specific, had intact open reading frames (ORFs and could potentially encode functional proteins. Phylogenetic analyses of UrsusERV consensus protein sequences suggest that it is part of a pig, gibbon and koala retrovirus clade. The young age estimates and lineage specificity of the virus suggests UrsusERV is a recent cross species transmission from an unknown reservoir and places the viral group among the youngest of ERVs identified in mammals.

• Legume roots develop two types of lateral organs, lateral roots and nodules. Nodules develop as a result of a symbiotic interaction with rhizobia and provide a niche for the bacteria to fix atmospheric nitrogen for the plant. • The Arabidopsis NAC1 transcription factor is involved in lateral root formation, and is regulated post-transcriptionally by miRNA164 and by SINAT5-dependent ubiquitination. We analyzed in Medicago truncatula the role of the closest NAC1 homolog in lateral root formation and in nodulation. • MtNAC1 shows a different expression pattern in response to auxin than its Arabidopsis homolog and no changes in lateral root number or nodulation were observed in plants affected in MtNAC1 expression. In addition, no interaction was found with SINA E3 ligases, suggesting that post-translational regulation of MtNAC1 does not occur in M. truncatula. Similar to what was found in Arabidopsis, a conserved miR164 target site was retrieved in MtNAC1, which reduced protein accumulation of a GFP-miR164 sensor. Furthermore, miR164 and MtNAC1 show an overlapping expression pattern in symbiotic nodules, and overexpression of this miRNA led to a reduction in nodule number. • This work suggests that regulatory pathways controlling a conservedtranscription factor are complex and divergent between M. truncatula and Arabidopsis.

Full Text Available Abstract Background The specific genetic regulation of neural primordial cell determination is of great interest in stem cell biology. The Musashi1 (Msi1 protein, which belongs to an evolutionarilyconserved family of RNA-binding proteins, is a marker for neural stem/progenitor cells (NS/PCs in the embryonic and post-natal central nervous system (CNS. Msi1 regulates the translation of its downstream targets, including m-Numb and p21 mRNAs. In vitro experiments using knockout mice have shown that Msi1 and its isoform Musashi2 (Msi2 keep NS/PCs in an undifferentiated and proliferative state. Msi1 is expressed not only in NS/PCs, but also in other somatic stem cells and in tumours. Based on previous findings, Msi1 is likely to be a key regulator for maintaining the characteristics of self-renewing stem cells. However, the mechanisms regulating Msi1 expression are not yet clear. Results To identify the DNA region affecting Msi1 transcription, we inserted the fusion gene ffLuc, comprised of the fluorescent Venus protein and firefly Luciferase, at the translation initiation site of the mouse Msi1 gene locus contained in a 184-kb bacterial artificial chromosome (BAC. Fluorescence and Luciferase activity, reflecting the Msi1 transcriptional activity, were observed in a stable BAC-carrying embryonic stem cell line when it was induced toward neural lineage differentiation by retinoic acid treatment. When neuronal differentiation was induced in embryoid body (EB-derived neurosphere cells, reporter signals were detected in Msi1-positive NSCs and GFAP-positive astrocytes, but not in MAP2-positive neurons. By introducing deletions into the BAC reporter gene and conducting further reporter experiments using a minimized enhancer region, we identified a region, "D5E2," that is responsible for Msi1 transcription in NS/PCs. Conclusions A regulatory element for Msi1 transcription in NS/PCs is located in the sixth intron of the Msi1 gene. The 595-bp D5E2 intronic

The mitochondrial DNA control region of the sun bear (Helarctos malayanus) was sequenced using 21 DNA samples collected from confiscated sun bears to identify conservation units, such as evolutionarily significant units and management units, in Sarawak, Borneo Island. A total of 10 haplotypes were observed, indicating the presence of at least two lineages in the sun bear population in Sarawak. Presumably, these two lineages could represent evolutionarily significant units. However, the geographical distributions of the two lineages remained unknown due to the lack of information regarding the exact capture locations of the confiscated sun bears. It is essential to elucidate the geographical distributions of these lineages in order to create a proper conservation plan for the sun bears in Sarawak. Therefore, further studies examining the haplotype distributions using DNA samples from known localities are essential.

Transcription of eukaryotic protein-coding genes commences with the assembly of a conserved initiation complex, which consists of RNA polymerase II (Pol II) and the general transcription factors, at promoter DNA. After two decades of research, the structural basis of transcription initiation is emerging. Crystal structures of many components of the initiation complex have been resolved, and structural information on Pol II complexes with general transcription factors has recently been obtained. Although mechanistic details await elucidation, available data outline how Pol II cooperates with the general transcription factors to bind to and open promoter DNA, and how Pol II directs RNA synthesis and escapes from the promoter.

Transcription represents the first step in gene expression. It is therefore not surprising that transcription is a highly regulated process and its control is essential to understand the flow and processing of information required by the cell to maintain its homeostasis. During transcription, a DNA molecule is copied into RNA molecules that are then used to translate the genetic information into proteins; this logical pattern has been conserved throughout all three kingdoms of life, from Arch...

Enhancers are traditionally viewed as DNA sequences located some distance from a promoter that act in cis and in an orientation-independent fashion to increase utilization of specific promoters and thereby regulate gene expression. Much progress has been made over the last decade toward understanding how these distant elements interact with target promoters, but how transcription is enhanced remains an object of active inquiry. Recent reports convey the prevalence and diversity of enhancer transcription and transcripts and support both as key factors with mechanistically distinct, but not mutually exclusive roles in enhancer function. Decoupling the causes and effects of transcription on the local chromatin landscape and understanding the role of enhancer transcripts in the context of long-range interactions are challenges that require additional attention. In this review, we focus on the possible functions of enhancer transcription by highlighting several recent enhancer RNA papers and, within the context of other enhancer studies, speculate on the role of enhancer transcription in regulating differential gene expression.

Full Text Available Short interspersed repetitive elements (SINEs are highly repeated sequences that account for a significant proportion of many eukaryotic genomes and are usually considered "junk DNA". However, we previously discovered that many AmnSINE1 loci are evolutionarilyconserved across mammalian genomes, suggesting that they may have acquired significant functions involved in controlling mammalian-specific traits. Notably, we identified the AS021 SINE locus, located 390 kbp upstream of Satb2. Using transgenic mice, we showed that this SINE displays specific enhancer activity in the developing cerebral cortex. The transcription factor Satb2 is expressed by cortical neurons extending axons through the corpus callosum and is a determinant of callosal versus subcortical projection. Mouse mutants reveal a crucial function for Sabt2 in corpus callosum formation. In this study, we compared the enhancer activity of the AS021 locus with Satb2 expression during telencephalic development in the mouse. First, we showed that the AS021 enhancer is specifically activated in early-born Satb2(+ neurons. Second, we demonstrated that the activity of the AS021 enhancer recapitulates the expression of Satb2 at later embryonic and postnatal stages in deep-layer but not superficial-layer neurons, suggesting the possibility that the expression of Satb2 in these two subpopulations of cortical neurons is under genetically distinct transcriptional control. Third, we showed that the AS021 enhancer is activated in neurons projecting through the corpus callosum, as described for Satb2(+ neurons. Notably, AS021 drives specific expression in axons crossing through the ventral (TAG1(-/NPY(+ portion of the corpus callosum, confirming that it is active in a subpopulation of callosal neurons. These data suggest that exaptation of the AS021 SINE locus might be involved in enhancement of Satb2 expression, leading to the establishment of interhemispheric communication via the corpus callosum

Full Text Available TORC1 is a central regulator of cell growth in response to amino acid availability, yet little is known about how it is regulated. Here, we performed a reverse genetic screen in yeast for genes necessary to inactivate TORC1. The screen consisted of monitoring the expression of a TORC1 sensitive GFP-based transcriptional reporter in all yeast deletion strains using flow cytometry. We find that in response to amino acid starvation, but not to carbon starvation or rapamycin treatment, cells lacking NPR2 and NPR3 fail to fully (1 activate transcription factors Gln3/Gat1, (2 dephosphorylate TORC1 effector Npr1, and (3 repress ribosomal protein gene expression. Both mutants show proliferation defects only in media containing a low quality nitrogen source, such as proline or ammonia, whereas no defects are evident when cells are grown in the presence of glutamine or peptone mixture. Proliferation defects in npr2Delta and npr3Delta cells can be completely rescued by artificially inhibiting TORC1 by rapamycin, demonstrating that overactive TORC1 in both strains prevents their ability to adapt to an environment containing a low quality nitrogen source. A biochemical purification of each demonstrates that Npr2 and Npr3 form a heterodimer, and this interaction is evolutionarilyconserved since the human homologs of NPR2 and NPR3 (NPRL2 and NPRL3, respectively also co-immunoprecipitate. We conclude that, in yeast, the Npr2/3 complex mediates an amino acid starvation signal to TORC1.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, and its occurrence is associated with a number of environmental factors including ingestion of the dietary contaminant aflatoxin B(1) (AFB(1)). Research over the last 40 years has revealed rainbow trout (Oncorhynchus mykiss) to be an excellent research model for study of AFB(1)-induced hepatocarcinogenesis; however, little is known about changes at the molecular level in trout tumors. We have developed a rainbow trout oligonucleotide array containing 1672 elements representing over 1400 genes of known or probable relevance to toxicology, comparative immunology, carcinogenesis, endocrinology, and stress physiology. In this study, we applied microarray technology to examine gene expression of AFB(1)-induced HCC in the rainbow trout tumor model. Carcinogenesis was initiated in trout embryos with 50 ppb AFB(1), and after 13 months control livers, tumors, and tumor-adjacent liver tissues were isolated from juvenile fish. Global gene expression was determined in histologically confirmed HCCs compared to noncancerous adjacent tissue and sham-initiated control liver. We observed distinct gene regulation patterns in HCC compared to noncancerous tissue including upregulation of genes important for cell cycle control, transcription, cytoskeletal formation, and the acute phase response and downregulation of genes involved in drug metabolism, lipid metabolism, and retinol metabolism. Interestingly, the expression profiles observed in trout HCC are similar to the transcriptional signatures found in human and rodent HCC, further supporting the validity of the model. Overall, these findings contribute to a better understanding of the mechanism of AFB(1)-induced hepatocarcinogenesis in trout and identify conserved genes important for carcinogenesis in species separated evolutionarily by more than 400 million years.

Autophagy is an evolutionarilyconserved degradative process that allows cells to maintain homoeostasis in numerous physiological situations. This process also functions as an essential protective response to endoplasmic reticulum (ER) stress, which promotes the removal and degradation of unfolded proteins. However, little is known regarding the mechanism by which autophagy is initiated and regulated in response to ER stress. In this study, different types of autophagy were identified in human gastric cancer MKN45 cells in response to the stress induced by nutrient starvation or lipotoxicity in which the regulation of these pathways is mammalian target of rapamycin (mTOR)-dependent or -independent, respectively. Interestingly, we found that p8, a stress-inducible transcription factor, was enhanced in MKN45 cells treated with palmitic acid to induce lipotoxicity. Furthermore, an increase in autophagy was observed in MKN45 cells stably overexpressing p8 using a lentivirus system, and autophagy induced by palmitic acid was blocked by p8 RNAi compared with the control. Western blotting analyses showed that autophagy was regulated by p8 or mTOR in response to the protein kinase-like endoplasmic reticulum kinase/activating transcription factor 6-mediated ER stress of lipotoxicity or the parkin-mediated mitochondrial stress of nutrient starvation, respectively. Furthermore, our results indicated that autophagy induced by palmitic acid is mTOR-independent, but this autophagy pathway was regulated by p8 via p53- and PKCα-mediated signaling in MKN45 cells. Our findings provide insights into the role of p8 in regulating autophagy induced by the lipotoxic effects of excess fat accumulation in cells.

Many Archaea, in contrast to bacteria, produce a high proportion of leaderless transcripts, show a wide variation in their consensus Shine-Dalgarno (S-D) sequences and frequently use GUG and UUG start codons. In order to understand the basis for these differences, 18 complete archaeal genomes were...... examined for sequence signals that are positionally conserved upstream from genes. These functional motifs include box A promoter sequences for leaderless transcripts and S-D sequences for transcripts with leaders. Most of the box A sequences were preceded by a BRE-like motif and followed by a previously...... for the 18 Archaea, reveal that usage of high levels of both S-D motifs, and GUG and UUG start codons occurs exclusively in the shorter branched Archaea. High levels of leaderless transcripts are found in the longer branched Archaea....

The yeast Set2 histone methyltransferase is a critical enzyme that plays a number of key roles in gene transcription and DNA repair. Recently, the human homologue, SETD2, was found to be recurrently mutated in a significant percentage of renal cell carcinomas, raising the possibility that the activity of SETD2 is tumor-suppressive. Using budding yeast and human cell line model systems, we examined the functional significance of two evolutionarilyconserved residues in SETD2 that are recurrently mutated in human cancers. Whereas one of these mutations (R2510H), located in the Set2 Rpb1 interaction domain, did not result in an observable defect in SETD2 enzymatic function, a second mutation in the catalytic domain of this enzyme (R1625C) resulted in a complete loss of histone H3 Lys-36 trimethylation (H3K36me3). This mutant showed unchanged thermal stability as compared with the wild type protein but diminished binding to the histone H3 tail. Surprisingly, mutation of the conserved residue in Set2 (R195C) similarly resulted in a complete loss of H3K36me3 but did not affect dimethylated histone H3 Lys-36 (H3K36me2) or functions associated with H3K36me2 in yeast. Collectively, these data imply a critical role for Arg-1625 in maintaining the protein interaction with H3 and specific H3K36me3 function of this enzyme, which is conserved from yeast to humans. They also may provide a refined biochemical explanation for how H3K36me3 loss leads to genomic instability and cancer.

In addition to its crucial role in seed production, pollen serves as a vector for gene flow between plant populations. Recently, pollen was identified as a mechanism for introduction of transgenes into non-transgenic populations. To investigate the genetic basis for pollen fitn...

In addition to its crucial role in seed production, pollen serves as a vector for gene flow between plant populations. Recently, pollen was identified as a mechanism for introduction of transgenes into non-transgenic populations. To investigate the genetic basis for pollen fitn...

Full Text Available Germins (GERs and germin-like proteins (GLPs play important roles in responses to various stresses; however, their function is still not fully understood. Significant insight into their function can be obtained by analyzing their promoters. In the present study, the 5' upstream promoters (1000 bp of 43 Asian rice (Oryza sativa var. Japonica GLP genes were retrieved from the Plant Ensemble, based on the Rice Annotation Project database (RAP-DB. Phylogenetic analysis via MEGA6 showed a narrow genetic background (0.2% with a Tajima neutrality value (π of 0.69. Overall, 4234 transcription factor (TF binding sites (TFBSs were found on chromosomes 1, 2, 3, 4, 5, 8, 9, 11 and 12 via “MatInspector” from 90 different TF families using a total of 444 families. Common TFs and DiAlign analyses showed that arabidopsis homeobox protein (AHBP, MYB-like proteins (MYBL and vertebrate TATA-box-binding protein (VTBP were the most abundant, common and evolutionarilyconserved elements in the upstream region from 0 to -800. Finding their mutual interaction via Farmworker analysis uncovered three new cisregulatory modules (VTBP_VTBP, MYBS_MYBS, and AHBP_VTBP, which appear to be decisive for OsGLP regulation. In silico functional analysis via ModelInspector revealed 77 cis-regulatory modules, each comprised of two elements, among which DOFF_OPAQ_03 and GTBX_MYCL_01 were the most frequent and mostly found on chromosome 8 and 12, indicating that the combinatorial interaction of these elements has a fundamental role in various biological processes. The study revealed the importance of these elements in regulating OsGLP expression that will help in predicting the role of these genes in various stresses, and can have application in biotechnology.

Full Text Available Polycomb (PcG and trithorax (trxG proteins play important roles in establishing lineage-specific genetic programs through induction of chromatin modifications that lead to gene silencing or activation. Previously, we described an association between the MLL/SET1 complexes and a highly restricted, gene-specific DNA-binding protein Ap2delta that is required for recruitment of the MLL/SET1 complex to target Hoxc8 specifically. Here, we reduced levels of Ap2delta and Ash2l in the neuroblastoma cell line, Neuro2A, and analyzed their gene expression profiles using whole-genome mouse cDNA microarrays. This analysis yielded 42 genes that are potentially co-regulated by Ap2delta and Ash2l, and we have identified evolutionarilyconserved Ap2-binding sites in 20 of them. To determine whether some of these were direct targets of the Ap2delta-Ash2l complex, we analyzed several promoters for the presence of Ap2delta and Ash2l by chromatin immunoprecipitation (ChIP. Among the targets we screened, we identified Fgfr3 as a direct transcriptional target of the Ap2delta-Ash2l complex. Additionally, we found that Ap2delta is necessary for the recruitment of Ash2l-containing complexes to this promoter and that this recruitment leads to trimethylation of lysine 4 of histone H3 (H3K4me3. Thus, we have identified several candidate targets of complexes containing Ap2delta and Ash2l that can be used to further elucidate their roles during development and showed that Fgfr3 is a novel direct target of these complexes.

Fungus-farming ant colonies vary four to five orders of magnitude in size. They employ compounds from actinomycete bacteria and exocrine glands as antimicrobial agents. Atta colonies have millions of ants and are particularly relevant for understanding hygienic strategies as they have abandoned their ancestors' prime dependence on antibiotic-based biological control in favour of using metapleural gland (MG) chemical secretions. Atta MGs are unique in synthesizing large quantities of phenylacetic acid (PAA), a known but little investigated antimicrobial agent. We show that particularly the smallest workers greatly reduce germination rates of Escovopsis and Metarhizium spores after actively applying PAA to experimental infection targets in garden fragments and transferring the spores to the ants' infrabuccal cavities. In vitro assays further indicated that Escovopsis strains isolated from evolutionarily derived leaf-cutting ants are less sensitive to PAA than strains from phylogenetically more basal fungus-farming ants, consistent with the dynamics of an evolutionary arms race between virulence and control for Escovopsis, but not Metarhizium. Atta ants form larger colonies with more extreme caste differentiation relative to other attines, in societies characterized by an almost complete absence of reproductive conflicts. We hypothesize that these changes are associated with unique evolutionary innovations in chemical pest management that appear robust against selection pressure for resistance by specialized mycopathogens. PMID:25925100

Transcription, the first step of gene expression, is carried out by the enzyme RNA polymerase (RNAP) and is regulated through interaction with a series of protein transcription factors. RNAP and its associated transcription factors are highly conserved across the bacterial domain and represent excellent targets for broad-spectrum antibacterial agent discovery. Despite the numerous antibiotics on the market, there are only two series currently approved that target transcription. The determination of the three-dimensional structures of RNAP and transcription complexes at high resolution over the last 15 years has led to renewed interest in targeting this essential process for antibiotic development by utilizing rational structure-based approaches. In this review, we describe the inhibition of the bacterial transcription process with respect to structural studies of RNAP, highlight recent progress toward the discovery of novel transcription inhibitors, and suggest additional potential antibacterial targets for rational drug design.

The SRY gene functions as a genetic switch in gonadal ridge initiating testis determination. The mouse Sry and human SRY open reading frames (ORFs) share a conserved DNA-binding domain (the HMG-box) yet exhibit no additional homology outside this region. As judged by the accumulation of lacZ-SRY hybrid proteins in the nucleus, both the human and mouse SRY ORFs contain a nuclear localization signal. The mouse Sry HMG-box domain selectively binds the sequence NACAAT in vitro when challenged with a random pool of oligonucleotides and binds AACAAT with the highest affinity. When put under the control of a heterologous promotor, the mouse Sry gene activated transcription of a reporter gene containing multiple copies of the AACAAT binding site. Activation was likewise observed for a GAL4-responsive reporter gene, when the mouse Sry gene was linked to the DNA-binding domain of GAL4. Using this system, the activation function was mapped to a glutamine/histidine-rich domain. In addition, LexA-mouse Sry fusion genes activated a LexA-responsive reporter gene in yeast. In contrast, a GAL4-human SRY fusion gene did not cause transcriptional activation. These studies suggest that both the human and the mouse SRY ORFs encode nuclear, DNA-binding proteins and that the mouse Sry ORF can function as a transcriptional activator with separable DNA-binding and activator domains.

Using the sequences of all the known transcription-associated proteins from Bacteria and Eucarya (a total of 4,147), we have identified their homologous counterparts in the four complete archaeal genomes. Through extensive sequence comparisons, we establish the presence of 280 predicted transcription factors or transcription-associated proteins in the four archaeal genomes, of which 168 have homologs only in Bacteria, 51 have homologs only in Eucarya, and the remaining 61 have homologs in both phylogenetic domains. Although bacterial and eukaryotic transcription have very few factors in common, each exclusively shares a significantly greater number with the Archaea, especially the Bacteria. This last fact contrasts with the obvious close relationship between the archaeal and eukaryotic transcription mechanisms per se, and in particular, basic transcription initiation. We interpret these results to mean that the archaeal transcription system has retained more ancestral characteristics than have the transcription mechanisms in either of the other two domains.

Alternative splicing represents a source of great diversity for regulating protein expression and function. It has been estimated that one-third to two-thirds of mammalian genes are alternatively spliced. With the sequencing of the chicken genome and analysis of transcripts expressed in chicken tissues, we are now in a position to address evolutionary conservation of alternative splicing events in chicken and mammals. Here, we compare chicken and mammalian transcript sequences of 41 alternatively-spliced genes and 50 frequently accessed genes. Our results support a high frequency of splicing events in chicken, similar to that observed in mammals. PMID:17675855

The highly conserved condensin complex is essential for the condensation and integrity of chromosomes through cell division. Published data argue that high levels of transcription contribute to specify some condensin-binding sites on chromosomes but the exact role of transcription in this process remains elusive. Here we discuss our recent data addressing the role of transcription in establishing a condensin-binding site.

The NRL gene encodes an evolutionarilyconserved basic motif-leucine zipper transcription factor that is implicated in regulating the expression of the photoreceptor-specific gene rhodopsin. NRL is expressed in postmitotic neuronal cells and in lens during embryonic development, but exhibits a retina-specific pattern of expression in the adult. To understand regulation of NRL expression and to investigate its possible involvement in retinopathies, we have determined the complete sequence of the human NRL gene, identified a polymorphic (CA)n repeat (identical to D14S64) within the NRL-containing cosmid, and refined its location by linkage analysis. Since a locus for autosomal recessive retinitis pigmentosa (arRP) has been linked to markers at 14q11 and since mutations in rhodopsin can lead to RP, we sequenced genomic PCR products of the NRL gene and of the rhodopsin-Nrl response element from a panel of patients representing independent families with inherited retinal degeneration. The analysis did not reveal any causative mutations in this group of patients. These investigations provide the basis for delineating the DNA sequence elements that regulate NRL expression in distinct neuronal cell types and should assist in the analysis of NRL as a candidate gene for inherited diseases/syndromes affecting visual function. Copyright 1997 Academic Press.

Plant-defense responses are triggered by perception of conserved microbe-associated molecular patterns (MAMPs), for example, flagellin or peptidoglycan. However, it remained unknown whether plants can detect conserved molecular patterns derived from plant-parasitic animals, including nematodes. Here we show that several genera of plant-parasitic nematodes produce small molecules called ascarosides, an evolutionarilyconserved family of nematode pheromones. Picomolar to micromolar concentrations of ascr#18, the major ascaroside in plant-parasitic nematodes, induce hallmark defense responses including the expression of genes associated with MAMP-triggered immunity, activation of mitogen-activated protein kinases, as well as salicylic acid- and jasmonic acid-mediated defense signalling pathways. Ascr#18 perception increases resistance in Arabidopsis, tomato, potato and barley to viral, bacterial, oomycete, fungal and nematode infections. These results indicate that plants recognize ascarosides as a conserved molecular signature of nematodes. Using small-molecule signals such as ascarosides to activate plant immune responses has potential utility to improve economic and environmental sustainability of agriculture.

The Mediator complex is an important component of the eukaryotic transcriptional machinery. As an essential link between transcription factors and RNA polymerase II, the Mediator complex transduces diverse signals to genes involved in different pathways. The plant Mediator complex was recently purified and comprises conserved and specific subunits. It functions in concert with transcription factors to modulate various responses. In this review, we summarize the recent advances in understanding the plant Mediator complex and its diverse roles in plant growth, development, defense, non-coding RNA production, response to abiotic stresses, flowering, genomic stability and metabolic homeostasis. In addition, the transcription factors interacting with the Mediator complex are also highlighted.

The completion of the Arabidopsis thaliana genome sequence allows a comparative analysis of transcriptional regulators across the three eukaryotic kingdoms. Arabidopsis dedicates over 5% of its genome to code for more than 1500 transcription factors, about 45% of which are from families specific to plants. Arabidopsis transcription factors that belong to families common to all eukaryotes do not share significant similarity with those of the other kingdoms beyond the conserved DNA binding domains, many of which have been arranged in combinations specific to each lineage. The genome-wide comparison reveals the evolutionary generation of diversity in the regulation of transcription.

Anthropogenic environmental impacts can disrupt the sensory environment of animals and affect important processes from mate choice to predator avoidance. Currently, these effects are best understood for auditory and chemosensory modalities, and recent reviews highlight their importance for conservation. We examined how anthropogenic changes to the visual environment (ambient light, transmission, and backgrounds) affect visual communication and camouflage and considered the implications of these effects for conservation. Human changes to the visual environment can increase predation risk by affecting camouflage effectiveness, lead to maladaptive patterns of mate choice, and disrupt mutualistic interactions between pollinators and plants. Implications for conservation are particularly evident for disrupted camouflage due to its tight links with survival. The conservation importance of impaired visual communication is less documented. The effects of anthropogenic changes on visual communication and camouflage may be severe when they affect critical processes such as pollination or species recognition. However, when impaired mate choice does not lead to hybridization, the conservation consequences are less clear. We suggest that the demographic effects of human impacts on visual communication and camouflage will be particularly strong when human-induced modifications to the visual environment are evolutionarily novel (i.e., very different from natural variation); affected species and populations have low levels of intraspecific (genotypic and phenotypic) variation and behavioral, sensory, or physiological plasticity; and the processes affected are directly related to survival (camouflage), species recognition, or number of offspring produced, rather than offspring quality or attractiveness. Our findings suggest that anthropogenic effects on the visual environment may be of similar importance relative to conservation as anthropogenic effects on other sensory modalities

Hot on the heels of last year's Sustainable Development Goals and the Paris Agreement, representatives from the global conservation community met to set the conservation agenda that will help to implement these targets.

When animals are traded in lucrative international luxury markets, individuals really do matter to conservation. Identifying the intrinsic and extrinsic factors that make some species especially vulnerable to this kind of threat helps set guidelines for more effective conservation.

Full Text Available The Golgi complex performs a central function in the secretory pathway in the sorting and sequential processing of a large number of proteins destined for other endomembrane organelles, the plasma membrane, or secretion from the cell, in addition to lipid metabolism and signaling. The Golgi apparatus can be regarded as a self-organizing system that maintains a relatively stable morphofunctional organization in the face of an enormous flux of lipids and proteins. A large number of the molecular players that operate in these processes have been identified, their functions and interactions defined, but there is still debate about many aspects that regulate protein trafficking and, in particular, the maintenance of these highly dynamic structures and processes. Here, we consider how an evolutionarilyconserved underlying mechanism based on retrograde trafficking that uses lipids, COPI, SNAREs, and tethers could maintain such a homeodynamic system.

Traditional explicit numerical discretizations of conservative systems generically predict artificial secular drifts of any nonlinear invariants. In this work the authors present a general approach for developing explicit nontraditional algorithms that conserve such invariants exactly. They illustrate the method by applying it to the three-wave truncation of the Euler equations, the Lotka-Volterra predator-prey model, and the Kepler problem. The ideas are discussed in the context of symplectic (phase-space-conserving) integration methods as well as nonsymplectic conservative methods. They comment on the application of the method to general conservative systems.

Traditional numerical discretizations of conservative systems generically yield an artificial secular drift of any nonlinear invariants. In this work we present an explicit nontraditional algorithm that exactly conserves invariants. We illustrate the general method by applying it to the Three-Wave truncation of the Euler equations, the Volterra-Lotka predator-prey model, and the Kepler problem. We discuss our method in the context of symplectic (phase space conserving) integration methods as well as nonsymplectic conservative methods. We comment on the application of our method to general conservative systems.

Traditional numerical discretizations of conservative systems generically yield an artificial secular drift of any nonlinear invariants. In this work we present an explicit nontraditional algorithm that exactly conserves these invariants. We illustrate the general method by applying it to the three-wave truncation of the Euler equations, the Lotka--Volterra predator--prey model, and the Kepler problem. This method is discussed in the context of symplectic (phase space conserving) integration methods as well as nonsymplectic conservative methods. We comment on the application of our method to general conservative systems.

Traditional numerical discretizations of conservative systems generically yield an artificial secular drift of any nonlinear invariants. In this work we present an explicit nontraditional algorithm that exactly conserves invariants. We illustrate the general method by applying it to the Three-Wave truncation of the Euler equations, the Volterra-Lotka predator-prey model, and the Kepler problem. We discuss our method in the context of symplectic (phase space conserving) integration methods as well as nonsymplectic conservative methods. We comment on the application of our method to general conservative systems.

Highly conserved short sequences help identify functional genomic regions and facilitate genomic annotation. We used Salmonella as the model to search the genome for evolutionarilyconserved regions and focused on the tetranucleotide sequence CTAG for its potentially important functions. In Salmonella, CTAG is highly conserved across the lineages and large numbers of CTAG-containing short sequences fall in intergenic regions, strongly indicating their biological importance. Computer modeling demonstrated stable stem-loop structures in some of the CTAG-containing intergenic regions, and substitution of a nucleotide of the CTAG sequence would radically rearrange the free energy and disrupt the structure. The postulated degeneration of CTAG takes distinct patterns among Salmonella lineages and provides novel information about genomic divergence and evolution of these bacterial pathogens. Comparison of the vertically and horizontally transmitted genomic segments showed different CTAG distribution landscapes, with the genome amelioration process to remove CTAG taking place inward from both terminals of the horizontally acquired segment. PMID:28262684

Highly conserved short sequences help identify functional genomic regions and facilitate genomic annotation. We used Salmonella as the model to search the genome for evolutionarilyconserved regions and focused on the tetranucleotide sequence CTAG for its potentially important functions. In Salmonella, CTAG is highly conserved across the lineages and large numbers of CTAG-containing short sequences fall in intergenic regions, strongly indicating their biological importance. Computer modeling demonstrated stable stem-loop structures in some of the CTAG-containing intergenic regions, and substitution of a nucleotide of the CTAG sequence would radically rearrange the free energy and disrupt the structure. The postulated degeneration of CTAG takes distinct patterns among Salmonella lineages and provides novel information about genomic divergence and evolution of these bacterial pathogens. Comparison of the vertically and horizontally transmitted genomic segments showed different CTAG distribution landscapes, with the genome amelioration process to remove CTAG taking place inward from both terminals of the horizontally acquired segment.

Microbes live in changing environments and change their phenotype via gene regulation in response. Although this transcriptional response is important for fitness, very little is known about how it evolves in microbes. We started by asking a number of high-level questions about the evolution of transcriptional phenotype: (1) To what extent is transcriptional response conserved, i.e. do conserved genes respond similarly to the same condition; (2) To what extent are transcriptional modules conserved; and (3) Does there exist a general stress response to a variety of stressors? To illuminate these questions, we analyzed more than 500 microarray experiments across the bacterial domain. We looked for conservation of transcriptional regulation both in close sister species and vastly divergent clades. In addition, we produced and analyzed an extensive in-house compendium of environmental stress data in three metal-reducing bacteria.

GPR177 is an evolutionarilyconserved transmembrane protein necessary for Wnt protein secretion. Little is currently known, however, regarding expression of GPR177, especially in vertebrate species. We have developed an antiserum against GPR177, and used it to examine expression of GPR177 in human tissue culture cells, adult mouse and rat tissues, as well as developing zebrafish embryos. In rodents, GPR177 is expressed in virtually all tissue types and brain regions examined. In zebrafish, GP...

Full Text Available Abstract Background Genomic imprinting is an evolutionary conserved mechanism of epigenetic gene regulation in placental mammals that results in silencing of one of the parental alleles. In order to decipher interactions between allele-specific DNA methylation of imprinted genes and evolutionary conservation, we performed a genome-wide comparative investigation of genomic sequences and highly conserved elements of imprinted genes in human and mouse. Results Evolutionarilyconserved elements in imprinted regions differ from those associated with autosomal genes in various ways. Whereas for maternally expressed genes strong divergence of protein-encoding sequences is most prominent, paternally expressed genes exhibit substantial conservation of coding and noncoding sequences. Conserved elements in imprinted regions are marked by enrichment of CpG dinucleotides and low (TpG+CpA/(2·CpG ratios indicate reduced CpG deamination. Interestingly, paternally and maternally expressed genes can be distinguished by differences in G+C and CpG contents that might be associated with unusual epigenetic features. Especially noncoding conserved elements of paternally expressed genes are exceptionally G+C and CpG rich. In addition, we confirmed a frequent occurrence of intronic CpG islands and observed a decelerated degeneration of ancient LINE-1 repeats. We also found a moderate enrichment of YY1 and CTCF binding sites in imprinted regions and identified several short sequence motifs in highly conserved elements that might act as additional regulatory elements. Conclusions We discovered several novel conserved DNA features that might be related to allele-specific DNA methylation. Our results hint at reduced CpG deamination rates in imprinted regions, which affects mostly noncoding conserved elements of paternally expressed genes. Pronounced differences between maternally and paternally expressed genes imply specific modes of evolution as a result of differences in

Full Text Available The Hox clusters play a crucial role in body patterning during animal development. They encode both Hox transcription factor and micro-RNA genes that are activated in a precise temporal and spatial sequence that follows their chromosomal order. These remarkable collinear properties confer functional unit status for Hox clusters. We developed the TranscriptView platform to establish high resolution transcriptional profiling and report here that transcription in the Hox clusters is far more complex than previously described in both human and mouse. Unannotated transcripts can represent up to 60% of the total transcriptional output of a cluster. In particular, we identified 14 non-coding Transcriptional Units antisense to Hox genes, 10 of which (70% have a detectable mouse homolog. Most of these Transcriptional Units in both human and mouse present conserved sizeable sequences (>40 bp overlapping Hox transcripts, suggesting that these Hox antisense transcripts are functional. Hox clusters also display at least seven polycistronic clusters, i.e., different genes being co-transcribed on long isoforms (up to 30 kb. This work provides a reevaluated framework for understanding Hox gene function and dys-function. Such extensive transcriptions may provide a structural explanation for Hox clustering.

Full Text Available BACKGROUND: Habitat loss and overexploitation are among the primary factors threatening populations of many mammal species. Recently, aquatic mammals have been highlighted as particularly vulnerable. Here we test (1 if aquatic mammals emerge as more phylogenetically urgent conservation priorities than their terrestrial relatives, and (2 if high priority species are receiving sufficient conservation effort. We also compare results among some phylogenetic conservation methods. METHODOLOGY/PRINCIPAL FINDINGS: A phylogenetic analysis of conservation priorities for all 620 species of Cetartiodactyla and Carnivora, including most aquatic mammals. Conservation priority ranking of aquatic versus terrestrial species is approximately proportional to their diversity. However, nearly all obligated freshwater cetartiodactylans are among the top conservation priority species. Further, ∼74% and 40% of fully aquatic cetartiodactylans and carnivores, respectively, are either threatened or data deficient, more so than their terrestrial relatives. Strikingly, only 3% of all 'high priority' species are thought to be stable. An overwhelming 97% of these species thus either show decreasing population trends (87% or are insufficiently known (10%. Furthermore, a disproportional number of highly evolutionarily distinct species are experiencing population decline, thus, such species should be closely monitored even if not currently threatened. Comparison among methods reveals that exact species ranking differs considerably among methods, nevertheless, most top priority species consistently rank high under any method. While we here favor one approach, we also suggest that a consensus approach may be useful when methods disagree. CONCLUSIONS/SIGNIFICANCE: These results reinforce prior findings, suggesting there is an urgent need to gather basic conservation data for aquatic mammals, and special conservation focus is needed on those confined to freshwater. That

Full Text Available The identification of homologies, whether morphological, molecular, or genetic, is fundamental to our understanding of common biological principles. Homologies bridging the great divide between deuterostomes and protostomes have served as the basis for current models of animal evolution and development. It is now appreciated that these two clades share a common developmental toolkit consisting of conservedtranscription factors and signaling pathways. These patterning genes sometimes show common expression patterns and genetic interactions, suggesting the existence of similar or even conserved regulatory apparatus. However, previous studies have found no regulatory sequence conserved between deuterostomes and protostomes. Here we describe the first such enhancers, which we call bilaterian conserved regulatory elements (Bicores. Bicores show conservation of sequence and gene synteny. Sequence conservation of Bicores reflects conserved patterns of transcription factor binding sites. We predict that Bicores act as response elements to signaling pathways, and we show that Bicores are developmental enhancers that drive expression of transcriptional repressors in the vertebrate central nervous system. Although the small number of identified Bicores suggests extensive rewiring of cis-regulation between the protostome and deuterostome clades, additional Bicores may be revealed as our understanding of cis-regulatory logic and sample of bilaterian genomes continue to grow.

activation of luciferase activity with the wildtype but not the mutant construct in all stages. These data suggest that Xenopus SET is able to bind to specific DNA sequences to activate transcription at all stages of Xenopus oogenesis. These data indicate that SET is an evolutionarilyconservedtranscription factor that participates in the early ontogenesis of the gonadal system, regulates P450c17 gene transcription in Leydig cells, and may also activate other genes expressed in immature oocytes, thus playing a role in oocyte development.

Cilia play many essential roles in fluid transport and cellular locomotion, and as sensory hubs for a variety of signal transduction pathways. Despite having a conserved basic morphology, cilia vary extensively in their shapes and sizes, ultrastructural details, numbers per cell, motility patterns and sensory capabilities. Emerging evidence indicates that this diversity, which is intimately linked to the different functions that cilia perform, is in large part programmed at the transcriptional level. Here, we review our understanding of the transcriptional control of ciliary biogenesis, highlighting the activities of FOXJ1 and the RFX family of transcriptional regulators. In addition, we examine how a number of signaling pathways, and lineage and cell fate determinants can induce and modulate ciliogenic programs to bring about the differentiation of distinct cilia types.

Full Text Available Regulation of transcription involves dynamic rearrangements of chromatin structure. The budding yeast Saccharomyces cerevisiae has a variety of highly conserved factors necessary for these reconstructions. Chromatin remodelers, histone modifiers and histone chaperones directly associate to promoters and open reading frames of exposed genes and facilitate activation and repression of transcription. We compare two distinct patterns of induced transcription: Sustained transcribed genes switch to an activated state where they remain as long as the induction signal is present. In contrast, single pulsed transcribed genes show a quick and strong induction pulse resulting in high transcript levels followed by adaptation and repression to basal levels. We discuss intensively studied promoters and coding regions from both groups for their co-factor requirements during transcription. Interplay between chromatin restructuring factors and dynamic transcription is highly variable and locus dependent.

The term “transcriptional network” refers to the mechanism(s) that underlies coordinated expression of genes, typically involving transcription factors (TFs) binding to the promoters of multiple genes, and individual genes controlled by multiple TFs. A multitude of studies in the last two decades have aimed to map and characterize transcriptional networks in the yeast Saccharomyces cerevisiae. We review the methodologies and accomplishments of these studies, as well as challenges we now face....

Transcription factors regulate the activities of RNA polymerase (RNAP) at each stage of the transcription cycle. Many basal transcription factors with common ancestry are employed in eukaryotic and archaeal systems that directly bind to RNAP and influence intramolecular movements of RNAP and modulate DNA or RNA interactions. We describe and employ a flexible methodology to directly probe and quantify the binding of transcription factors to RNAP in vivo. We demonstrate that binding of the conserved and essential archaeal transcription factor TFE to the archaeal RNAP is directed, in part, by interactions with the RpoE subunit of RNAP. As the surfaces involved are conserved in many eukaryotic and archaeal systems, the identified TFE-RNAP interactions are likely conserved in archaeal-eukaryal systems and represent an important point of contact that can influence the efficiency of transcription initiation.

In eukaryotes, sequence-specific DNA-binding proteins activate gene expression by recruiting the transcriptional apparatus and chromatin remodeling proteins to the promoter through protein-protein contacts. In many instances, the connection between DNA-binding proteins and the transcriptional apparatus is established through the intermediacy of adapter proteins known as coactivators. Here we describe synthetic molecules with low molecular weight that act as transcriptional coactivators. We demonstrate that a completely nonnatural activation domain in one such molecule is capable of stimulating transcription in vitro and in vivo. The present strategy provides a means of gaining external control over gene activation through intervention using small molecules.

SUMMARY We report the development of a next-generation sequencing-based technology that entails construction of a DNA library comprising up to at least 47 (~16,000) bar-coded sequences, production of RNA transcripts, and analysis of transcript ends and transcript yields ("massively systematic transcript end readout," MASTER). Using MASTER, we define full inventories of transcription start sites ("TSSomes") of Escherichia coli RNA polymerase for initiation at a consensus core promoter in vitro and in vivo, we define the TSS-region DNA-sequence determinants for TSS selection, reiterative initiation ("slippage synthesis"), and transcript yield, and we define effects of DNA topology and NTP concentration. The results reveal that slippage synthesis occurs from the majority of TSS-region DNA sequences and that TSS-region DNA sequences have profound, up to 100-fold, effects on transcript yield. The results further reveal that TSSomes depend on DNA topology, consistent with the proposal that TSS selection involves transcription-bubble expansion ("scrunching") and transcription-bubble contraction ("anti-scrunching"). PMID:26626484

We report the development of a next-generation sequencing-based technology that entails construction of a DNA library comprising up to at least 4(7) (∼ 16,000) barcoded sequences, production of RNA transcripts, and analysis of transcript ends and transcript yields (massively systematic transcript end readout, "MASTER"). Using MASTER, we define full inventories of transcription start sites ("TSSomes") of Escherichia coli RNA polymerase for initiation at a consensus core promoter in vitro and in vivo; we define the TSS-region DNA sequence determinants for TSS selection, reiterative initiation ("slippage synthesis"), and transcript yield; and we define effects of DNA topology and NTP concentration. The results reveal that slippage synthesis occurs from the majority of TSS-region DNA sequences and that TSS-region DNA sequences have profound, up to 100-fold, effects on transcript yield. The results further reveal that TSSomes depend on DNA topology, consistent with the proposal that TSS selection involves transcription-bubble expansion ("scrunching") and transcription-bubble contraction ("anti-scrunching").

Plant-specific transcription factor NAC proteins play essential roles in many biological processes such as development, senescence, morphogenesis, and stress signal transduction pathways. In the NAC family, some members function as transcription activators while others act as repressors. In the present study we found that though the full-length GmNAC20 from soybean did not have transcriptional activation activity, the carboxy-terminal activation domain of GmNAC20 had high transcriptional activation activity in the yeast assay system. Deletion experiments revealed an active repression domain with 35 amino acids, named NARD (NAC Repression Domain), in the d subdomain of NAC DNA-binding domain. NARD can reduce the transcriptional activation ability of diverse transcription factors when fused to either the amino-terminal or the carboxy-terminal of the transcription factors. NARD-like sequences are also present in other NAC family members and they are functional repression domain when fused to VP16 in plant protoplast assay system. Mutation analysis of conserved amino acid residues in NARD showed that the hydrophobic LVFY motif may partially contribute to the repression function. It is hypothesized that the interactions between the repression domain NARD and the carboxy-terminal activation domain may finally determine the ability of NAC family proteins to regulate downstream gene expressions.

The following aspects of energy conservation were discussed: conservation history and goals, conservation modes, conservation accounting-criteria, and a method to overcome obstacles. The conservation modes tested fall into one of the following categories: reduced energy consumption, increased efficiency of energy utilization, or substitution of one or more forms of energy for another which is in shorter supply or in some sense thought to be of more value. The conservation accounting criteria include net energy reduction, economic, and technical criteria. A method to overcome obstacles includes (approaches such as: direct personal impact (life style, income, security, aspiration), an element of crisis, large scale involvement of environmental, safety, and health issues, connections to big government, big business, big politics, involvement of known and speculative science and technology, appeal to moral and ethical standards, the transient nature of opportunities to correct the system.

Leaf mass per area (LMA) and leaf lifespan (LL) are two highly correlated plant traits that are key to plant physiological and ecological properties. Usually, low LMA means short LL, high nitrogen (N) content per unit mass, and fast turnover rates of nutrients; high LMA leads to long LL, low N content, and slow turnover rates. Deciduous trees with low LMA and short lifespan leaves have low carbon cost but high nitrogen demand; and evergreen trees, with high LMA and long lifespan leaves, have high carbon cost but low nitrogen demand. These relationships lead to: 1) evergreen trees have higher leaf area index than deciduous trees; 2) evergreen trees' carbon use efficiency is lower than the deciduous trees' because of their thick leaves and therefore high maintenance respiration; 3) the advantage of evergreens trees brought by their extra leaves over deciduous trees diminishes with increase N in ecosystem. These facts determine who will win when trees compete with each other in a N-limited ecosystem. In this study, we formulate a mathematical model according to the relationships between LMA, LL, leaf nitrogen, and leaf building and maintenance cost, where LMA is the fundamental variable determining the other three. We analyze the evolutionarily stable strategies (ESSs) of LMA with this mathematical model by examining the benefits of carbon and nitrogen investments to leaves in ecosystems with different N. The model shows the ESS converges to low LMA at high N and high LMA at low N. At intermediate N, there are two ESSs at low and high ends of LMA, respectively. The ESS also leads to low forest productivity by outcompeting the possible high productive strategies. We design a simulation scheme in an individual-based competition model (LM3-PPA) to simulate forest dynamics as results of the competition between deciduous and evergreen trees in three different biomes, which are temperate deciduous forest, deciduous-evergreen mixed forest, and boreal evergreen forest. The

Full Text Available Abstract Background The exponential growth of the number of fully sequenced genomes at varying taxonomic closeness allows one to characterize transcriptional regulation using comparative-genomics analysis instead of time-consuming experimental methods. A transcriptional regulatory unit consists of a transcription factor, its binding site and a regulated gene. These units constitute a graph which contains so-called “network motifs”, subgraphs of a given structure. Here we consider genomes of closely related Enterobacteriales and estimate the fraction of conserved network motifs and sites as well as positions under selection in various types of non-coding regions. Results Using a newly developed technique, we found that the highest fraction of positions under selection, approximately 50%, was observed in synvergon spacers (between consecutive genes from the same strand, followed by ~45% in divergon spacers (common 5’-regions, and ~10% in convergon spacers (common 3’-regions. The fraction of selected positions in functional regions was higher, 60% in transcription factor-binding sites and ~45% in terminators and promoters. Small, but significant differences were observed between Escherichia coli and Salmonella enterica. This fraction is similar to the one observed in eukaryotes. The conservation of binding sites demonstrated some differences between types of regulatory units. In E. coli, strains the interactions of the type “local transcriptional factor gene” turned out to be more conserved in feed-forward loops (FFLs compared to non-motif interactions. The coherent FFLs tend to be less conserved than the incoherent FFLs. A natural explanation is that the former imply functional redundancy. Conclusions A naïve hypothesis that FFL would be highly conserved turned out to be not entirely true: its conservation depends on its status in the transcriptional network and also from its usage. The fraction of positions under selection in

Full Text Available Population fragmentation is threatening biodiversity worldwide. Species that once roamed vast areas are increasingly being conserved in small, isolated areas. Modern management approaches must adapt to ensure the continued survival and conservation value of these populations. In South Africa, a managed metapopulation approach has been adopted for several large carnivore species, all protected in isolated, relatively small, reserves that are fenced. As far as possible these approaches are based on natural metapopulation structures. In this network, over the past 25 years, African lions (Panthera leo were reintroduced into 44 fenced reserves with little attention given to maintaining genetic diversity. To examine the situation, we investigated the current genetic provenance and diversity of these lions. We found that overall genetic diversity was similar to that in a large national park, and included a mixture of four different southern African evolutionarily significant units (ESUs. This mixing of ESUs, while not ideal, provides a unique opportunity to study the impact of mixing ESUs over the long term. We propose a strategic managed metapopulation plan to ensure the maintenance of genetic diversity and improve the long-term conservation value of these lions. This managed metapopulation approach could be applied to other species under similar ecological constraints around the globe.

Full Text Available Abstract Background Gene expression measurements from breast cancer (BrCa tumors are established clinical predictive tools to identify tumor subtypes, identify patients showing poor/good prognosis, and identify patients likely to have disease recurrence. However, diverse breast cancer datasets in conjunction with diagnostic clinical arrays show little overlap in the sets of genes identified. One approach to identify a set of consistently dysregulated candidate genes in these tumors is to employ meta-analysis of multiple independent microarray datasets. This allows one to compare expression data from a diverse collection of breast tumor array datasets generated on either cDNA or oligonucleotide arrays. Results We gathered expression data from 9 published microarray studies examining estrogen receptor positive (ER+ and estrogen receptor negative (ER- BrCa tumor cases from the Oncomine database. We performed a meta-analysis and identified genes that were universally up or down regulated with respect to ER+ versus ER- tumor status. We surveyed both the proximal promoter and 3' untranslated regions (3'UTR of our top-ranking genes in each expression group to test whether common sequence elements may contribute to the observed expression patterns. Utilizing a combination of known transcription factor binding sites (TFBS, evolutionarilyconserved mammalian promoter and 3'UTR motifs, and microRNA (miRNA seed sequences, we identified numerous motifs that were disproportionately represented between the two gene classes suggesting a common regulatory network for the observed gene expression patterns. Conclusion Some of the genes we identified distinguish key transcripts previously seen in array studies, while others are newly defined. Many of the genes identified as overexpressed in ER- tumors were previously identified as expression markers for neoplastic transformation in multiple human cancers. Moreover, our motif analysis identified a collection of

The enteric NtrC (NRI) protein has been the paradigm for a class of bacterial enhancer-binding proteins (EBPs) that activate transcription of RNA polymerase containing the sigma 54 factor. Activators in the NtrC class are characterized by essentially three properties: (i) they bind to sites distant from the promoters that they activate (> 100 bp upstream of the transcriptional start site), (ii) they contain a conserved nucleotide-binding fold and exhibit ATPase activity that is required for a...

Over the past twenty years conservation genetics has progressed from being mainly a theory-based field of population biology to a full-grown empirical discipline. Technological developments in molecular genetics have led to extensive use of neutral molecular markers such as microsatellites in conser

ABSTRACT: Here we present the Transcription Factor Encyclopedia (TFe), a new web-based compendium of mini review articles on transcription factors (TFs) that is founded on the principles of open access and collaboration. Our consortium of over 100 researchers has collectively contributed over 130...

The application of new and less biased methods to study the transcriptional output from genomes, such as tiling arrays and deep sequencing, has revealed that most of the genome is transcribed and that there is substantial overlap of transcripts derived from the two strands of DNA. In protein codi...

Parasitic species, which depend directly on host species for their survival, represent a major regulatory force in ecosystems and a significant component of Earth's biodiversity. Yet the negative impacts of parasites observed at the host level have motivated a conservation paradigm of eradication, moving us farther from attainment of taxonomically unbiased conservation goals. Despite a growing body of literature highlighting the importance of parasite-inclusive conservation, most parasite species remain understudied, underfunded, and underappreciated. We argue the protection of parasitic biodiversity requires a paradigm shift in the perception and valuation of their role as consumer species, similar to that of apex predators in the mid-20th century. Beyond recognizing parasites as vital trophic regulators, existing tools available to conservation practitioners should explicitly account for the unique threats facing dependent species. We built upon concepts from epidemiology and economics (e.g., host-density threshold and cost-benefit analysis) to devise novel metrics of margin of error and minimum investment for parasite conservation. We define margin of error as the risk of accidental host extinction from misestimating equilibrium population sizes and predicted oscillations, while minimum investment represents the cost associated with conserving the additional hosts required to maintain viable parasite populations. This framework will aid in the identification of readily conserved parasites that present minimal health risks. To establish parasite conservation, we propose an extension of population viability analysis for host-parasite assemblages to assess extinction risk. In the direst cases, ex situ breeding programs for parasites should be evaluated to maximize success without undermining host protection. Though parasitic species pose a considerable conservation challenge, adaptations to conservation tools will help protect parasite biodiversity in the face of

The mechanical properties of transcription have recently been shown to play a central role in gene expression. However, a full physical characterization of this central biological process is lacking. In this Letter, we introduce a simple description of the basic physical elements of transcription where RNA elongation, RNA polymerase rotation, and DNA supercoiling are coupled. The resulting framework describes the relative amount of RNA polymerase rotation and DNA supercoiling that occurs during RNA elongation. Asymptotic behavior is derived and can be used to experimentally extract unknown mechanical parameters of transcription. Mechanical limits to transcription are incorporated through the addition of a DNA supercoiling-dependent RNA polymerase velocity. This addition can lead to transcriptional stalling and resulting implications for gene expression, chromatin structure and genome organization are discussed.

A transcriptional activator can suppress gene expression by interfering with transcription initiated by another activator. Transcriptional interference has been increasingly recognized as a regulatory mechanism of gene expression. The signals received by the two antagonistically acting activators are combined by the polymerase trafficking along the DNA. We have designed a dual-control genetic system in yeast to explore this antagonism systematically. Antagonism by an upstream activator bears the hallmarks of competitive inhibition, whereas a downstream activator inhibits gene expression non-competitively. When gene expression is induced weakly, the antagonistic activator can have a positive effect and can even trigger paradoxical activation. Equilibrium and non-equilibrium models of transcription shed light on the mechanism by which interference converts signals, and reveals that self-antagonism of activators imitates the behavior of feed-forward loops. Indeed, a synthetic circuit generates a bell-shaped response, so that the induction of expression is limited to a narrow range of the input signal. The identification of conserved regulatory principles of interference will help to predict the transcriptional response of genes in their genomic context. PMID:19690569

A nationwide examination was made of grassroots energy conservation programs related to transportation. Information compiled from civic groups, trade associations, and corporations is included on driver awareness/mass transit; travel; and ride sharing. It is concluded that a willingness by the public to cooperate in transportation energy conservation exists and should be exploited. (LCL)

Take plant lessons outdoors with this engaging and inquiry-based activity in which third-grade students learn how to apply soil conservation methods to growing plants. They also collect data and draw conclusions about the effectiveness of their method of soil conservation. An added benefit to this activity is that the third-grade students played…

Asian's remarkable economic growth brought many benefits but also fuelled threats to its ecosystems and biodiversity. Economic growth brings biodiversity threats but also conservation opportunities. Continued biodiversity loss is inevitable, but the types, areas and rates of biodiversity loss are not. Prioritising biodiversity conservation, tempered by what is tractable, remains a high priority. Policy and market distortions and failures significantly underprice biodiversity, undermine ecosys...

Alarmed by the growing water shortage, the New Jersey State Office of Dissemination has prepared this annotated list of free or inexpensive instructional materials for teaching about water conservation, K-l2. A tipsheet for home water conservation is appended. (Editor/SJL)

Included are ideas, suggestions, and examples for selecting and designing conservation science projects. Over 70 possible conservation subject areas are presented with suggested projects. References are cited with each of these subject areas, and a separate list of annotated references is included. The references pertain to general subject…

This glossary is a composite of terms selected from 13 technologies, and is the expanded revision of the original 1952 edition of "The Soil and Water Conservation Glossary." The terms were selected from these areas: agronomy, biology, conservation, ecology, economics, engineering, forestry, geology, hydrology, range, recreation, soils, and…